Cardiac and perivascular myofibroblasts, matrifibrocytes, and immune fibrocytes in hypertension; commonalities and differences with other cardiovascular diseases

Abstract

Hypertension is a major cause of cardiovascular diseases such as myocardial infarction and stroke. Cardiovascular fibrosis occurs with hypertension and contributes to vascular resistance, aortic stiffness, and cardiac hypertrophy. However, the molecular mechanisms leading to fibroblast activation in hypertension remain largely unknown. There are two types of fibrosis: replacement fibrosis and reactive fibrosis. Replacement fibrosis occurs in response to the loss of viable tissue to form a scar. Reactive fibrosis occurs in response to an increase in mechanical and neurohormonal stress. Although both types of fibrosis are considered adaptive processes, they become maladaptive when the tissue loss is too large, or the stress persists. Myofibroblasts represent a subpopulation of activated fibroblasts that have gained contractile function to promote wound healing. Therefore, myofibroblasts are a critical cell type that promotes replacement fibrosis. Although myofibroblasts were recognized as the fibroblasts participating in reactive fibrosis, recent experimental evidence indicated there are distinct fibroblast populations in cardiovascular reactive fibrosis. Accordingly, we will discuss the updated definition of fibroblast subpopulations, the regulatory mechanisms, and their potential roles in cardiovascular pathophysiology utilizing new knowledge from various lineage tracing and single-cell RNA sequencing studies. Among the fibroblast subpopulations, we will highlight the novel roles of matrifibrocytes and immune fibrocytes in cardiovascular fibrosis including experimental models of hypertension, pressure overload, myocardial infarction, atherosclerosis, aortic aneurysm, and nephrosclerosis. Exploration into the molecular mechanisms involved in the differentiation and activation of those fibroblast subpopulations may lead to novel treatments for end-organ damage associated with hypertension and other cardiovascular diseases.

Hypertension, Fibroblast, Fibrosis, Pressure overload, Myocardial Infarction

1. Introduction

Hypertension, a major cause of cardiovascular disease, is estimated to be prevalent in 45∼46% of the adult population in countries like United States and China.¹ Less than 14% of these patients are adequately controlled despite the development of improved treatments.² Hypertension is directly associated with many adverse events, including ischaemic heart disease, stroke, and kidney disease.² Hypertension causes cardiac perivascular and interstitial fibrosis leading to arterial stiffness and diastolic dysfunction along with myocardial hypertrophy.³ In experimental models of cardiac pressure overload, fibroblast expansion and activation were observed.⁴ Activated fibroblasts differentiate into myofibroblasts, a large cell type essential for wound healing, which produces structural extracellular matrix (ECM) proteins⁵ and express several contractile proteins including Acta2-encoded smooth muscle actin alpha 2 (αSMA).⁶ Myofibroblasts, a kind of activated fibroblast with migratory and contractile phenotype, are distinguishable from quiescent fibroblasts by their ruffled membranes, stress fibres, high level of exocytotic vesicles, and bundles of microfilaments.⁷ The primary function of myofibroblasts is to repair damaged tissue, ultimately contributing to the formation of a collagen scar. Classically, it was believed that quiescent tissue-resident fibroblasts are activated and differentiate into myofibroblasts in response to mechanical stress and inflammatory mediators.⁷ However, formalizing a molecular definition for myofibroblasts is a challenging task due to a lack of definitive cellular markers⁸ and the associated conceptual and hypothetical features. Indeed, myofibroblasts are frequently defined just as activated fibroblasts.⁹ Myofibroblast re-definition in cardiovascular pathophysiology is, therefore, one of the main purposes of this review and will be comprehensively discussed in the main text.

According to the three recent major review articles^9–11 and a milestone publication¹² regarding cardiac fibrosis and fibroblasts, myofibroblasts have been defined as one of the four subpopulations of fibroblasts. These subpopulations include resting/quiescent basal stage fibroblasts, proliferating/pre-activated stage fibroblasts, myofibroblasts/activated fibroblasts, and fibroblasts in resolution stage including matrifibrocytes.^9–11 The term, matrifibrocyte, was proposed to define the αSMA negative, matrix producing, nonproliferative fibroblast subpopulation derived from myofibroblasts found in the stable scar.¹² Matrifibrocytes preferentially express markers of bone, connective tissue, cartilage, and tendon development and appear important to maintain the scar integrity to preserve cardiac functions.¹² A common characteristic of all four stages is the expression of collagens. Periostin, a ligand for integrins to mediate cell adhesion and migration, is expressed in multiple, but not all fibroblast stages, whereas a smooth muscle marker protein, αSMA, is selectively expressed in one stage^9–11 (Figure 1A). However, these stages have been primarily defined during cardiac fibrosis in response to experimental myocardial infarction (MI) and are therefore considered model- and context-dependent. Unique to MI, the fibrotic response after MI involves two types of fibrosis, replacement fibrosis and reactive fibrosis.⁸ The replacement fibrosis (cicatricial scar formation) is a critical process to prevent ventricle rupture by replacing dead cardiac tissue with fibrosis. Myofibroblast induction seems particularly important for this replacement fibrosis since contractile myofibers may be needed to replace the large wound and prevent cardiac rupture upon MI.⁸ Reactive fibrosis includes interstitial and perivascular fibrosis and occurs as an adaptive response to mechanical as well as neurohormonal stress seen in hypertension and cardiac pressure overload.¹³ Thus, these stages may not be applicable to more chronic types of cardiovascular fibrosis such as those associated with hypertension, atherosclerosis, and chronic kidney disease.

Figure 1

Fibroblast subpopulations and their markers seen upon MI. (A) Simplified 4 fibroblast stage proposal in response to MI. Around days 2∼4 post MI, proliferation and migration of fibroblast populations are observed. ECM production is not enhanced. These proliferating fibroblasts expressing periostin will differentiate to myofibroblasts at the infract zone on day 4∼7. Myofibroblasts are the main fibroblast population contributing to the cardiac scar formation. These fibroblasts gain contractility and actively produce ECM components to complete replacement fibrosis. Upon completion of the scar, myofibroblasts are believed to further differentiate to matrifibrocytes to maintain the scar. However, cardiac mechanical stress and neurohormonal stress persist due to cardiac dysfunction caused by MI and insufficient compensation by the scar, leading to reactive fibrosis. Matrifibrocytes produce ECM components and contribute to reactive fibrosis as an adaptation to chronic cardiac injury. Marker genes and proteins preferentially expressed in each stage have been reported. Resting fibroblasts can be identified by the fibroblast markers as discussed. Proliferating fibroblasts expressing stem cell markers including Ly6A/Sca1, activated fibroblast marker periostin, cell cycle genes, inflammatory cytokines, and chemokines demonstrate their pro-fibrotic pro-inflammatory roles in the initiation of cardiac inflammation and fibrosis. Both myofibroblasts and matrifibrocytes highly express ECM genes and are involved in early replacement fibrosis and chronic reactive fibrosis, respectively. Myofibroblasts express periostin and contractile genes including Acta2/αSMA. Matrifibrocytes localize to scar site and border zones and express genes related to bone and cartilage remodelling, such as Comp, Chad, and Cilp1/2. Italics indicate the marker genes. (B) Updated understanding of fibroblast subpopulations and their markers seen upon MI. Resident resting fibroblasts appear to be the major source of fibroblasts involved in scar formation and reactive fibrosis upon MI. Three IR fibroblasts are the first responders to MI and cause an acute pro-inflammatory response at the site. Myofibroblast subpopulations proliferate and are actively involved in matrix production, scar formation and inflammatory conditions. At days 14∼28, these fibroblasts differentiate to matrifibrocytes, late resolution fibroblasts, and fibrocytes. These cell types play major roles in chronic interstitial fibrosis and inflammation as adaptation to post MI cardiac dysfunction. It is also important to note that some matrifibrocytes and other late stage fibroblasts may show stress-induced senescence and senescence-associated secretory phenotype/SASP, thus accelerate cardiac remodelling. Abbreviations are Act-fib; activated fibroblast; fib; fibroblast, Myofib; myofibroblast. Italics indicate the marker genes.

Another factor potentially influencing the fate of fibroblasts is that myofibroblasts can be differentiated from many lineages not just from resting fibroblasts. Accordingly, it is important to note that researchers may use the term myofibroblast to minimally define active fibroblasts in cardiovascular disease,^14–16 likely due to the difficulties with defining the fibroblast population. Therefore, in this review article, we will try to define the stage of fibroblasts, the marker and lineage of fibroblast subpopulations seen in hypertension, and how the myofibroblast features overlap or are distinct from those seen in post-MI and other major models of cardiovascular fibrosis.

The acute wound repair by myofibroblasts is considered physiological. However, in several forms of cardiovascular disease, this process is believed to become maladaptive because the tissue damage or injury is persistent and chronic. Cardiovascular fibrosis is characterized by excessive production and deposition of ECM caused by myofibroblast differentiation, and activation is associated with hypertension.¹⁷ Typically, fibrosis associated with hypertension occurs in interstitial space of the heart and kidney as well as perivascular adventitia in the aorta, heart, and kidney.^18,19 Excessive cardiac interstitial fibrosis worsens life expectancy by decreasing ventricular diastolic capacity and increasing lethal arrhythmias.²⁰ Aortic adventitial fibrosis also contributes to aortic stiffness and exaggerates cardiac hypertrophy and hypertension.²¹ It is therefore very important to understand the origin of pathologically activated fibroblasts as well as defining their unique markers seen in hypertension. The ‘hypertensive fibroblasts’ can be differentiated from quiescent resident fibroblasts, resident mesenchymal stem cells (MSC) (discussed in detail later), or another remote cell type of distinct lineage in given anatomical locations (interstitial vs. adventitia in the tissues they express). This information is a critical first step in understanding the molecular mechanism of cardiovascular fibroblast differentiation and activation, which is essential to seek therapeutic interventions against hypertensive cardiovascular fibrosis.

2. Definition of myofibroblasts in hypertension

To determine if there is a consensus in the field for the definition of myofibroblast, the molecular mechanism of myofibroblast induction, and the functional relevance of myofibroblasts in hypertension, we performed an Ovid literature search using the words ‘hypertension’ and ‘myofibroblast’. Of the 284 references (October 2023) obtained, 81 were identified that specifically studied myofibroblasts in hypertension. Supplementary material online, Table S1, summarizes how these articles defined myofibroblasts. In 90.12% (73 of 81) of the articles, αSMA was used as the sole myofibroblast marker. Most of these articles used αSMA staining as a way to define differentiation of myofibroblasts in cardiovascular fibrosis and did not study the molecular mechanism of myofibroblast induction or myofibroblast contribution to hypertension. A concern about using αSMA as a myofibroblast marker is that it is also expressed in vascular smooth muscle cells (VSMCs) and pericytes. Consequently, it is difficult to define myofibroblast solely by αSMA since it may also track migrated VSMC. Major weaknesses in using αSMA to define myofibroblasts (or activated fibroblasts) will be discussed in this review. Unfortunately, publications that precisely track myofibroblasts under hypertensive conditions or explore their roles in organ dysfunction associated with hypertension are limited. Using the limited but relevant literature available, this review article will explore the cell lineage, potential regulatory mechanism of fibroblast activation in hypertension, contribution to cardiovascular remodeling, dysfunction associated with hypertension, and the possibility of translation of the information towards a treatment. We will also discuss updated characteristics and refinements in evaluation methods for fibroblast subpopulations including myofibroblasts in animal models for hypertension and other major cardiovascular diseases.

3. Fibroblasts and myofibroblasts in hearts

3.1 Activated cardiac fibroblasts and their markers in response to pressure overload

Numerous molecules have been identified as markers for cardiac fibroblasts (17 proteins listed in reference¹⁰). Examples include vimentin, a cytoskeletal intermediate filament protein, fibroblast-specific protein 1 (FSP1 encoded by S100A4),²² discoidin domain receptor tyrosine kinase 2 (DDR2, a collagen-activated receptor kinase),²³ and Thy-1 cell surface antigen [also known as cluster of differentiation-90 (CD90)].²⁴ These fibroblast markers are not specific to cardiac fibroblasts and are known to be expressed in other cell types, which has been a major challenge. For example, vimentin and CD90 are expressed in endothelial and immune cells; FSP1 is expressed in many other cell types¹⁰ (pericytes, VSMC, endothelial cells, and immune cells) with cardiac fibroblasts making up a small percentage of total FSP1 expressing cells.^17,25  S100A4/FSP1 promoter has been shown to lack specificity and cannot be used to identify cardiac fibroblast induction in post-MI or transverse aortic constriction (TAC).²² Thus gene-targeting by S100A4-Cre mice is unlikely to be fibroblast specific.¹⁴ However, there were about 15% FSP1-positive fibroblasts after MI. These fibroblasts appear to demonstrate pro-angiogenic properties and did not overlap with αSMA positive myofibroblasts.²⁶ Functionally, FSP1 is a significant contributor to fibrotic responses in lung²⁷ and kidney.²⁸ Therefore, FSP1 remains an important subject for fibrosis research.

Lineage tracing in transgenic mouse models has been recently adapted to identify and target fibroblasts and their precursors in conditions of tissue fibrosis. In this technique, a murine Col1a1 promoter-green fluorescent protein (GFP) fusion reporter that specifically labels collagen type I α1 producing cells is utilized to track cardiac fibroblast lineage. Under basal conditions, the GFP-expressing cardiac fibroblasts were vimentin-positive, and platelet-derived growth factor receptor α (PDGFRα, a mesenchymal cell marker) positive. However, these resident cardiac fibroblasts seen in interstitial and perivascular locations were αSMA negative, PDGFRβ (pericyte marker) low or negative, platelet and endothelial cell adhesion molecule 1 (PECAM1, endothelial marker) negative, and CD45 (leucocyte marker) negative. Notably these fibroblasts were also negative for FSP1, a frequently utilized marker for fibroblasts.²⁹ Upon TAC (days 7 and 28), GFP-positive cells accumulated at cardiac fibrosis areas including perivascular area and remained positive for PDGFRα and mostly negative for FSP1 and CD45. The cells in perivascular fibrosis area remained αSMA negative and most (∼85%) of cells in interstitial fibrosis area were αSMA negative. Additional lineage tracing experiments demonstrated that quiescent cardiac fibroblasts were either Wt1 (Wilms tumour 1 suppressor gene) positive lineage (∼80%, epicardium origin) or Tie2 positive lineage (∼20%, a subset of endothelial-mesenchymal transition/EndoMT occurring in endocardial cells).²⁹ Upon TAC, these cell types showed significant proliferation in the fibrosis area with no change in the ratio (Wt1/Tie2). Combination of Vav-Cre (VAV1 encoding haematopoietic cells), VEC-Cre (vascular endothelial cadherin promotor to track endothelial cells), or Wt1-Cre tracer mice with Col1a1-GFP fusion reporter demonstrated that increased fibroblast populations after TAC was not derived from haematopoietic cells, EndoMT, or epicardial EMT, but were mostly due to resident fibroblast proliferation.²⁹ One concern of this study is that there is no statement or discussion regarding myofibroblasts such as whether a slightly increased αSMA positive subpopulation of interstitial fibroblasts represents myofibroblasts. These authors, however, described in a review article that in a pathological context ‘activated fibroblasts’ express progenitor markers such as WT1, a transcriptional factor T-box 18, and transcription factor 21 (TCF21).^29,30 These three transcriptional factors are expressed in epicardium-derived cells during development but re-expressed in collagen-producing fibroblasts after MI.³¹ Among these markers only TCF21 was described to be upregulated in perivascular fibrosis upon angiotensin II (Ang II) infusion as well as TAC.³² However, all three markers were positive at interstitial fibrosis upon TAC or Ang II infusion.³²

Recently, dual recombinase-mediated genetic lineage tracing revealed that Nfatc1-Dre Col1a2-Cre dual positive endocardium-derived fibroblasts represent a significant portion of the population of proliferating fibroblasts upon TAC surgery.³³ This subpopulation was considered to be Tie2 positive fibroblasts that proliferated in response to TAC in the prior study.²⁹ Removal of these cells reduced cardiac fibrosis and preserved cardiac function after TAC treatment.³³

3.2 Myofibroblasts do not contribute to cardiac fibrosis following TAC or Ang II infusion

Kanisicak et al. performed a lineage tracing analysis using Postn (periostin) gene-reporter mice and multiple Cre-expressing mouse lines and proposed periostin, a matricellular protein and a ligand for αVβ3 and αVβ5 integrins, as a myofibroblast-specific marker.³⁴ Chronic Ang II plus phenylephrine (Ang II/PE) treatment induced Postn gene-reporter positive myofibroblasts throughout the heart which were co-stained with vimentin and αSMA. The study further demonstrated that this promoter is minimally expressed in endothelial, bone marrow (BM)-derived, or smooth muscle cell (SMC) lineages in the heart, but is derived from tissue-resident fibroblasts of the Tcf21 lineage in 3 distinct cardiac injury models (MI, TAC, and Ang II/PE).³⁴ Subsequent work from the group demonstrated that Tcf21-Cre mediated deletion of p38α in cardiac resident fibroblasts or Postn-Cre mediated myofibroblast p38α silencing significantly reduced cardiac fibrosis post MI or Ang II/PE treatment.³⁵ Transforming growth factor β (TGF-β) signalling through Type I and Type II receptors via mothers against decapentaplegic homolog-2/3 (Smad2/Smad3) transcriptional factor phosphorylation plays critical roles in tissue fibrosis. Tcf21-Cre as well as Postn-Cre mediated cardiac silencing of Tgfbr1/2 or Smad3 attenuated fibrosis and fibroblast DNA synthesis in TAC model.³⁶ In addition, a protein chaperone, heat shock protein 47 (Hsp47) is involved in procollagen biosynthesis. Tcf21-Cre as well as Postn-Cre mediated Hsp47 silencing attenuated cardiac fibrosis and collagen deposition in TAC model.³⁷ Recently, Postn-Cre mice were used to silence myofibroblast expression of Col1a2 gene. Inducible silencing of Col1a2 with Postn-Cre mice transiently prevented collagen I accumulation and cardiac hypertrophy in response to TAC likely due to several compensatory mechanisms including TGF-β activation.³⁸ However, up to this point we have discussed Postn-Cre mediated putative cardiac myofibroblast targeting studies based on the assumption that Postn-traced cardiac myofibroblasts are derived from Tcf21-expressing resident cardiac fibroblasts and contribute to pathological cardiac fibrosis under pressure stress.³⁵ A critical concern remains unanswered whether Postn-Cre targeting is selective against myofibroblasts. Accumulating evidence discussed below suggests that the answer may be ‘no’.

In studies where pathological cardiac fibrosis was induced in animal models with MI, TAC, or Ang II infusion, cardiac myofibroblasts have been suggested to originate from a variety of sources, including tissue-resident fibroblasts, perivascular cells, BM-derived progenitor cells, fibrocytes (myeloid cell lineage fibroblast-like cells), epithelial-mesenchymal transition (EMT), and endothelial-mesenchymal transition (EndoMT) (reviewed in³⁹). As in the prior hypertension studies listed in Supplementary material online, Table S1, many of these studies utilized αSMA as the main marker for myofibroblasts.³⁹ However, by comparing three recent publications utilizing single cell transcriptomics to look for changes in cardiac fibroblast sub-populations and their gene expression patterns, it is evident that αSMA encoded by Acta2 was enhanced only in the early phases of post-MI in mice⁴⁰ and no significant change in Acta2 expression in fibroblast subpopulations was observed upon TAC at day 14 or 28⁴¹ or with Ang II infusion for 2 weeks.⁴² Taken together with the Col1a1 lineage tracing study in the TAC model,²⁹ these data suggest that αSMA expressing myofibroblasts are unlikely to contribute to myocardial fibrosis seen in many hypertension models.

Glioma-associated oncogene (Gli) family zinc finger 1 (Gli1) which mediates sonic hedgehog signalling, was reported as a novel lineage tracer for MSC that differentiate from activated fibroblasts.⁴³  Gli1-reporter expressed in mice was able to track activated fibroblasts in cardiac interstitial fibrosis induced by TAC.⁴³ Some Gli1-positive cells were stained with αSMA in the fibrosis.⁴³ A recent comprehensive single cell RNA sequencing (scRNA-seq) study of tracer mice looking at 6 distinct cell types at two distinct time points (days 14 and 28 post-TAC), significantly advanced our understanding of the characteristics of active ECM producing fibroblasts under cardiac pressure stress.⁴¹ No evidence was obtained for fibroblast differentiation from other cell types tracked by vascular endothelial cadherin coding Cdh5- (endothelial cells), smooth muscle myosin heavy chain (SMMHC) coding Myh11- (SMCs and pericytes), or chondroitin sulphate proteoglycan-4 coding Ng2- (pericyte and neural cells). All three fibroblast-targeting Cre-drivers (Col1a1 to trace fibroblasts, Gli1 to trace MSC, and PDGFR β-coding Pdgfrb to trace fibroblasts and mural cells (VSMC and pericyte)) detected fibroblast clusters before and after TAC.⁴¹ Upon TAC no fibroblast subcluster gained Acta2 expression suggesting a lack of myofibroblast induction by TAC. The expressing fibroblasts (ECM-fib) subcluster of fibroblasts actively produces ECM components (Col1a1/2) and highly expresses Postn and cartilage oligomeric matrix protein-coding Comp. These cells were significantly increased at days 14 and 28 and they also preferentially express thrombospondin 4-encoding Thbs4.⁴¹ As with Ang II infusion⁴² no evidence of proliferating fibroblast subpopulation was obtained post-TAC, suggesting differentiation of ECM-fib from existing fibroblast populations. Three major fibroblast subpopulations were confirmed in published scRNA-seq data sets with Ang II infusion⁴² and post MI.⁴⁰ They were (i) fibroblasts with high ECM expression: ECM-fib,⁴¹ FibThbs4/FibCilp,⁴² matrifibrocytes/MFCs⁴⁰; (ii) interferon responsive Int-Fib, and (iii) Ly6a/CD248 positive progenitor-like state fibroblasts.^40–42 The number of Int-Fib cells transiently increased at day 14 and preferentially expressed Ly6a and interferon-induced protein with tetratricopeptide repeats 1/3 coding Ifit1 and Ifit3, whereas progenitor-like fibroblast populations remained stable during TAC.⁴¹  Table 1 summarizes cardiac fibroblast lineage tracing studies in mouse models of cardiac pressure overload (TAC) and neurohormonal stress (i.e. Ang II discussed in the following section).

Table 1

Cardiac fibroblast lineage tracing under hormonal and pressure stress

References	Model	Marker	Quiescent	Matrifibrocyte	Matrifibrocyte
Moore-Morris29				Day 7	Day 28
	TAC	Col1a1-αSMA		15% interstitiala	∼5%
		Col1a1-Wt1		∼80%	∼80%
McLellan42	Ang II		Saline	Day14	Day 28
		Thbs4	Low	High, Comp, Postn
		Cilp	Low	High, Comp, Postn
Kuar44	Ang II	Postn-LacZ	None		High
Kramann43	Ang II	Gli1- αSMA	None		High at adventitia
			Control	Day 14
Kanisicak34	Ang II PE	Postn-marker	Low	High, Vimentin, αSMA
Kanisicak34	TAC	Postn-marker	Low	High, Vimentin, αSMA
			Control		Day 56
Kramann43	TAC	Gli1-αSMA	None		High at interstitial
			Sham	Day 14
Peisker41	TAC	Pdgfrb-Thbs4	None	High at interstitial
		Gli1-Thbs4	None	High at interstitial

Italics indicate the marker genes.

αSMA, smooth muscle α actin; PE, phenylephrine; TAC, Transverse aortic constriction; Thbs4, thrombospondin 4.

^aCol1a1-αSMA positive cells were not observed at perivascular fibrosis.

3.3 Matrifibrocytes as important contributors to cardiac fibrosis induced by Ang II infusion

Studies from Dr. Entman’s group demonstrated that chronic Ang II infusion increases cardiac DDR2 and collagen type I ECM-Fib positive for a haematopoietic marker, CD45, and a haematopoietic stem cell marker, CD34. Ang II-induced increases in CD45/CD34 double-positive monocytic and mesenchymal fibroblasts and cardiac fibrosis were attenuated in monocyte chemoattractant protein 1 (MCP-1/Ccl2) knock out mice.⁴⁵ Subsequent studies demonstrated that infiltration of collagen I negative M1 cells occurred in the heart upon Ang II infusion for 3 days.⁴⁶ After a week of infusion, M1 cells were minimally detected but collagen type I stained cells were significantly increased with characteristics of CD301/CD45 positive and CD206/CD45 positive M2 cells suggesting the induction or recruitment of M2 immune fibrocytes. The lineage of these Ang II-induced cardiac immune cell-marked fibroblasts is considered to be activated fibrocytes similar to those seen in aortic adventitia upon Ang II infusion.⁴⁷ In general, inflammatory M1 macrophages kill pathogens and act in host defense, whereas alternative anti-inflammatory M2 macrophages repair and maintain tissue integrity.⁴⁸ Under fibrotic disease, however, M1 macrophages have been implicated in cytokine-mediated recruitment of fibrocytes to initiate fibrosis whereas subsequent M2 macrophages drive fibrosis via production of TGFβ which activates resident fibroblasts.⁴⁸ Presence of fibrocytes in normal mouse heart has been confirmed by scRNA-seq. The fibrocytes were identified as a macrophage subpopulation expressing both fibroblast (Col1a1, Pdgfra (a mesenchymal stem cell (MSC) marker), and Tcf21) and immune cell (high-affinity immunoglobulin γ Fc receptor I coding Fcgr1, Cd14, and leukocyte-common antigen/protein tyrosine phosphatase receptor type C coding Ptprc) markers.⁴⁹

Periostin was also identified as a reliable marker to detect activated cardiac fibroblasts in mice infused with Ang II. Digested cardiac fibroblasts from mouse hearts infused with 2 μg/kg/min for 6–10 days were enriched by 1 h culture dish attachment. Single cell qPCR was performed for cell lineage markers to identify Cdh5, Myh11, Myh6 (a cardiac myocyte marker), or Itgam (CD11b) positive endothelial cells, SMC, cardiac myocytes, or myeloid cells, which comprised 40% of the attached cells. These lineage-positive cells were excluded from the study and only Col1α1 positive cells were analyzed as putative cardiac fibroblasts.⁴⁴ Compared with cardiac fibroblasts purified from control mice, fibroblasts from Ang II-infused mice express more fibrotic genes (several collagens, Fn1 (fibronectin-1), Tgfb and Lox (lysyl oxidase)). Although Ang II infusion increased all fibroblast markers tested in these cells, Acta2 (αSMA), S100A4, or Thy1 were only expressed in 33, 32, and 53% of these fibroblasts under Ang II infusion. In contrast, Postn, Ddr2, and Pdgfra were expressed in 94, 88, and 86% of the cells. Moreover, expression levels of Postn correlated best to all fibrotic genes tested indicating that periostin expression may be the best marker for active ECM producing fibroblasts in hypertensive hearts. Based on these findings the authors created tamoxifen inducible Postn-Cre reporter mice. Postn-Cre expression was confirmed at the interstitial space of the cardiac wall upon Ang II infusion. Interestingly, induction of Postn-Cre was not observed in aorta or kidney with Ang II infusion⁴⁴ suggesting that periostin may be a specific marker for cardiac-activated fibroblasts (note that this assumption may not agree with other fibrosis models in arteries or kidney discussed in the later sections). Postn-Cre mediated removal of activated fibroblasts reduced cardiac fibrosis upon Ang II infusion or post-MI and improved cardiac functional parameters but had no effect on cardiac hypertrophy.⁴⁴

As mentioned, Ang II infusion for 2 weeks increased the populations of cardiac fibroblasts highly expressing cartilage intermediate layer protein-encoding Cilp (basal ∼2% compared to Ang II 2 weeks ∼15%) or Thbs4 (basal very low compared to Ang II 2 weeks ∼25%). Immunofluorescent staining showed that THBS4 expression was localized at interstitial fibrosis area but not at perivascular fibrosis area of the heart. Both cell types also preferentially expressed the matrifibrocyte markers (Comp and secreted frizzled-related protein 2 coding Sfrp2) as well as ECM remodeling genes and Postn but did not express Acta2/αSMA.⁴² Pseudotime analysis suggests that Thbs4 cells arose from Cilp cells. Thus, Ang II infusion differentiated quiescent cardiac fibroblasts to Postn-positive activated interstitial fibroblasts with matrifibrocyte features distinct from myofibroblasts induced upon MI. Overall, the critical fibroblast population actively participating in cardiac interstitial fibrosis upon pressure overload as well as Ang II-induced hypertension appears to be ECM-fib/matrifibrocytes. Characteristics of this cell type are illustrated in Figure 2. Whether there are transition stages such as pro-inflammatory and proliferative fibroblasts and myofibroblasts before the appearance of matrifibrocytes under cardiac pressure overload or Ang II-induced hypertension remains to be investigated.

Figure 2

Cardiac fibroblast subpopulations and their markers seen upon TAC or Ang II infusion. Significant increases in Col1a1/2 and Postn ECM-Fib occur days 14 and 28 post TAC. This population appears to represent Fibroblast-Cilp plus Fibroblast-Thbs4 populations seen upon Ang II infusion as well as matrifibrocytes upon MI. ECM-Fib cells do not express myofibroblast marker, Acta2. These fibroblasts are therefore the main mediator of cardiac interstitial fibrosis in response to enhanced afterload (TAC or Ang II hypertension). In addition, the transient increase in Interferon-Fib cells at 2 weeks post TAC suggests the role to be pro-inflammatory. The inflammatory fibroblasts express stem cell marker, Ly6A but do not proliferate. Fibroblast progenitor cells are also present with no change in the population upon TAC or Ang II infusion. Abbreviations are Ang II, angiotensin II; col, collagen; ECM-Fib, extracellular matrix fibroblasts; Int-fib, interferon-; TAC, Transverse aortic constriction. Italics indicate the marker genes.

3.4 Cardiac fibroblast fate upon MI

As mentioned in sections 3.2 and 3.3, myofibroblasts may not be required for interstitial fibrosis (reactive fibrosis) caused by increased afterload.⁴¹ Reactive fibrosis that occurs in the remote zone after MI may have similar aetiologies to reactive fibrosis that occurs during other cardiovascular disease, which involves cardiac mechanical stress and exposure to hormonal mediators such as Ang II and norepinephrine. However, reactive fibrosis that occurs in the border zone after MI is unique to MI and involves distinct mechanical stress and additional neurohormonal components such as acute inflammatory responses, ischaemia reperfusion, chronic hypoxia, and exposure to pro-angiogenic factors.⁸ The border zone is rich with αSMA expressing myofibroblasts co-stained with non-muscle myosin heavy chain B (SMemb coded by Myh10).⁵⁰ The border zone fibrosis is clinically important because of the arrhythmogenic potential of the border zone myocytes. Important determinants for the arrhythmogenic potential appear to be fibrotic paracrine and heterocellular junctions.³

To track the fate of activated fibroblasts at MI infarct sites over time, lineage tracking was performed with Tcf21 promoter (resident fibroblasts) or Postn promoter (activated fibroblasts) combined with EdU labelling to evaluate fibroblast proliferation or Acta2/αSMA tracking for myofibroblast induction.¹² This approach identified fibroblasts with four distinct subpopulations. They are quiescent fibroblasts, proliferating fibroblasts, myofibroblasts, and matrifibrocytes. Using the Tcf21 promoter, they labelled quiescent fibroblasts, which correspond to tissue-resident fibroblasts, and found that proliferatively fibroblasts (active fibroblasts) appeared 2–4 days after MI at the border zone, with the conversion of fibroblasts to myofibroblasts occurring 4–7 days after infarction. Based on this study, the marker proteins expressed in quiescent fibroblasts, active fibroblasts, and myofibroblasts upon MI are illustrated in Figure 1A. Seven to ten days after MI, both proliferative capacity and αSMA expression decreased, indicating a shift to cells that maintain an ECM (matrifibrocytes). Analysis of the expressed mRNAs suggested that cells in different states have different expression patterns. Matrifibrocytes are unique cells because they localize to scar sites and express genes associated with bone and cartilage remodelling, such as chondroadherin-coding Chad, cartilage intermediate layer protein-coding Cilp2, and Comp.¹² In this study, it was also shown that Tcf21 lineage fibroblasts did not express endothelial marker CD31 or leukocyte marker CD45 upon MI in any of the fibroblast stages. In addition, CD31 or CD45 did not stain Acta2-lineage cells at day 7 post MI.¹²

Activated fibroblasts also alter their phenotype in several ways. They can revert to resting fibroblasts, undergo apoptosis, or enter senescence.¹¹ Whether similar fates can be seen in any animal models of hypertension remains unclear. The Tcf21-Cre mice were also utilized to silence αSMA-encoding Acta2 to test for its role in myofibroblast differentiation upon MI. While the cardiac phenotype was not modified due to compensative expression of other actin isoforms,⁵¹ the data suggest that Acta2 is an important structural component of pathological induction of myofibroblast. Proliferating fibroblasts upon MI were also observed by using Ki67-knock in mice.⁵² In contrast to the prior study,¹² appearance of proliferating fibroblasts was higher at day 14 than day 3 or 7 and formed a fibrotic scar. Subclusters containing neonatal like injury-associated fibroblasts were increased upon MI and highly expressed Col1a2, Col3a1, bone-mineralization promoting osteonectin-coded Sparc, a cardiomyogenic factor, follistatin-like protein 1 coding Fstl1. Col1a1-mediated fibroblast Fstl1 deletion resulted in cardiac rupture upon MI suggesting the importance of proliferating cardiac fibroblast for replacement fibrosis upon MI.⁵²

Appearance of myofibroblasts at day 7 post-MI was also reported with scRNA-seq analysis performed on sham or MI hearts.⁵³ 11 fibroblast subpopulations were identified including 2 minor subpopulations (fibroblasts with macrophage or endothelial cell identity). The myofibroblasts were only detected at day 7 and represent ∼50% of Pdgfra-GFP positive and CD31 negative cardiac fibroblasts (1% in control normal hearts) and show high expression of numerous collagen genes, Postn (∼99%), and Acta2 (αSMA) (61%).⁵³ The myofibroblasts highly expressed fibronectin coding Fn and collagen triple helix repeat containing I coding Cthrc1, the latter representing a highly specific marker for the myofibroblasts post MI. These cells also showed decreased stem/progenitor cell fate. This study also confirmed a transient increase in pre-activated fibroblasts at day 3, representing 48% of Pdgfra-GFP positive fibroblasts post MI (sham 7% and day 7 12%). Please note that in this study these cells were named activated fibroblasts.⁵³ The activated fibroblasts expressed less Acta2 (28%) but more Ly6a. They also preferentially expressed a matricellular protein and inhibitor of TGF-β signalling, Cilp, consistent with a pre-myofibroblast population. While being dominant during the early time point, this subpopulation appears distinct from the proliferating fibroblasts. The proliferating fibroblast cluster (∼0% at control, 15% at day 3, and 3% at day 7 post MI of the total fibroblast population) was separately identified with strong expression of cell cycle genes and high levels of Postn and Acta2.⁵³

Recently new post-MI fibroblast subpopulations were reported by a scRNA-seq study performing comprehensive time course assays. Using mouse hearts carrying epicardial-derived cell reporter (Wt1-EGFP), the fate of post MI fibroblast clusters was analyzed at multiple time points (day 0, 1, 3, 5. 7, 14, and 28).⁴⁰ 11 relevant fibroblast clusters were identified. Injury response (IR) fibroblast cluster was transiently increased in the population immediately after MI and expressed genes involved in oxidative stress metallothionein-coding Mt1 and Mt2, pro-inflammatory chemokines (the chemokine (C-C motif) ligand 2 (CCL2)/MCP-1 coding Ccl2, Ccl7, the chemokine (C-X-C motif) ligand 1 coding Cxcl1). This subpopulation transitioned to myofibroblasts on day 3. From day 3 to day 7, three subclusters forming myofibroblasts (Cthrc1, Acta2, Postn) were dominant. The subclusters included myofibroblasts (Acta2, Cthrc1), proliferating myofibroblasts (Acta2, Cthrc1, cell-cycle genes), and myeloid-myofibroblasts (a mixture of the expression of myeloid cell and myofibroblast cell markers; β-actin-coding Actb and lysozyme 2-coding Lyz2). This study also confirmed the enrichment of matrifibrocytes with higher expression of ECM components at day 14 to day 28.⁴⁰ Matrifibrocytes were positively stained with Clusterin, a stress-induced senescent fibroblast marker at day 14–28.⁴⁰ In addition, from day 14 to day 28, increase in late-resolution fibroblast cluster was observed. This cluster expressed genes associated with cell differentiation, osteogenesis, and regulation of matrix remodelling and deposition including a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-like 2 coding Adamtls2, Cilp, Col8a1, and mesenchyme homeobox1 coding Meox1. Although a cluster of fibroblast progenitors was observed, no change was observed in this cluster during the post-MI.

Potential contribution of pericytes producing collagen and other ECM components has been suggested by a scRNA-seq study upon TAC in mice.⁴¹ NG2 proteoglycan is a well-established marker for pericytes. Recently, cardiac pericytes were shown to be differentiated from myofibroblasts upon MI. NG2/PDGFRα pericyte/fibroblast reporter mice were used to identify pericyte-derived myofibroblasts. These cells account for a small subpopulation (∼4%) of post MI fibroblasts, potentially mediate vascular maturation, and may not regulate fibrotic remodelling.⁵⁴ In fact, when pericyte and VSMC lineages (mural cells) were traced by T-box18 encoding Tbx18-promoter, no evidence was obtained that these cell types produce collagen using Col1a1-GFP double transgenic mice 28 days after TAC surgery.⁵⁵

In contrast to the above-referenced findings indicating no contribution of myeloid cell-derived activated fibroblasts in post MI cardiac fibrosis, an earlier study used lysozyme 2-coding Lyz2 tracer mice to track induction of myeloid cell-derived fibroblast-like cells (fibrocytes) upon MI. Co-staining with fibroblast markers [Col1a1, FSP1, and fibroblast activation protein-α (FAP)] but not a myofibroblast marker αSMA was observed at 1- and 6 weeks post MI.⁵⁶ Ability of macrophages to differentiate to collagen-producing fibroblasts in infarct hearts was also shown with adoptive transfer of collagen-GFP tracer spleen-derived macrophages.⁵⁷ Immune cell-derived activated fibrocytes appeared highest at day 7 post MI but remained elevated by half at day 28. Mac2 also known as galectin-3 was shown to be a better marker to track fibrocytes than Mac3 also known as lysosome-associated membrane protein 2 (LAMP2) or lysozyme 2. About 12% of Mac2-positive cells were fibrocytes at day 7.⁵⁸ These findings were indirectly supported by the infarct heart fibroblasts acquiring phagocytic properties.⁵⁹ It is also possible that fibrocytes were differentiated from the myeloid-myofibroblast subcluster during post-MI.

Table 2 summarizes cardiac fibroblast lineage tracing studies in mouse models of post-MI. Figure 1B illustrates updated understanding of fibroblast subpopulations upon MI. Overall, post-MI injury-responsive pro-inflammatory and proliferative fibroblast subpopulations provide the initial environment for inflammation and fibroblast proliferation, which leads to myofibroblast mediated scar formation. In the myofibroblast subpopulations, there are fibroblasts demonstrating a mixed myeloid fibroblast lineage. These immune myofibroblasts are the source of fibrocytes which promote chronic inflammatory fibrosis. Matrifibrocytes and late-resolution fibroblasts then maintain scar structure and ECM environments and contribute to border zone remodelling with interstitial fibrosis.

Table 2

Post MI cardiac fibroblast characterization by lineage tracing

References	Marker	Quiescent	Activated Fib	Myofibroblast	Matrifibrocyte
Fu12		Day 0	Days 2–4	Days 4–7	Day 10∼
	Sirius red	±	+	++	++++
	Tcf21-	+	++	++++	++++
	Tcf21-Ki67	Low	High	Low	Low
	Tcf21-αSMA	Low	High	High	Low
	Acta2-EdU		High	High	Low
	Tcf21-Col3	+	++	+++	+++
	Tcf21-Comp	Low		Low	high
	Tcf21-Chad	Low		Low	high
Forte40		Quiescent	Injury resp	3 Myofb	MFC & LR
		Day 0	Days 1–3	Days 3–7	Days 14–28
	Wt1-MT1/2	Low	High
	Col1-CTHRC1	Low	Low	High	low
	Col1-Clusterin			Low	high
		Before MI	MΦ	Fibrocytes/MΦ	Fibrocytes/MΦ
Chen58		Control	Day 3	Day 7	Day 28
	Pdgfa-Lyz2	–	+	++/+++	+/+
	Pdgfa-Mac2	Low	+	++++/+++	++/+
	Pdgfa-Mac3	Low	+	+++/++	+++/+
Haider56				Week 1	Week 6
	LysM-Col1A1			Low	High
	LysM-FSP1			Low	High
	LysM-FAP			High	Low
	LysM-αSMA			None	None
Alex54		Pericyte	Pericyte	NG2+ Myofb	NG2+ Myofb
		Control	Day 3	Day 7	Day 28
	NG2-αSMA			+++	+
	NG2-Pdgfra	Low		+++	++

Italics indicate the marker genes.

αSMA, smooth muscle α actin; Chad, chondroadherin; col3, collagen 3; Comp, cartilage oligomeric matrix protein; CTHRC1, collagen triple helix repeat containing 1; EdU, 5-ethynyl-2′-deoxyuridine; FAP, fibroblast activation protein-α; Fib, fibroblast; FSP1, fibroblast specific protein 1; Lyz2, lysozyme 2; Mac2, galectin-3; Mac3, lysosome-associated membrane protein 2; Mt1, metallothionein 1; NG2, chondroitin sulphate proteoglycan-4.

3.5 Cardiac fibroblast in aging, senescence, and premature senescence

Aged hearts demonstrate myocyte hypertrophy as well as diffuse interstitial fibrosis in the absence of any injury, which leads to stiffness of the heart and the development of cardiac dysfunction.^60,61 Accumulating evidence suggests that aging hearts are accompanied by pro-inflammatory and pro-fibrotic signalling in cardiac fibroblasts via autocrine and paracrine release of neurohormonal factors including Ang II and TGF-β. Changes in the ECM structure further cause mechanical alteration in intracellular signalling leading to proliferation of fibroblasts and differentiation to myofibroblasts.⁶² However, aged cardiac fibroblasts showed impaired mechanosensing and difficulty with myofibroblast conversion upon MI, which leads to reduced strength of the scar and sustained post-MI dysfunction with pathological remodelling.⁶³ Cellular mechanisms of aging associated cardiac fibrosis remain poorly understood. A recent study with scRNA-seq on young and old mouse hearts demonstrated that cardiac fibroblasts distribute to 11 different clusters. Among them, clusters enriched for genes associated with immune response and osteoblasts were increased in old mice.⁶⁴ An increase in the cluster expressing osteoblast genes suggests a reactive fibroblast differentiation to matrifibrocytes during aging.

Senescent fibroblasts are increased by aging and show hyper-secretory phenotype termed SASP (senescence-associated secretory phenotype).⁶⁵ SASP seems to be a significant contributor to pro-inflammatory and fibrotic cardiac environment associated with aging.⁶⁵ Premature senescence is senescence that occurs upon cellular stress and injury independently from chronological aging. A significant increase in premature senescent cells was observed in perivascular fibrotic area of the coronary arteries upon TAC. These cells were positive for αSMA and PDGFRα thus considered as myofibroblasts. Interestingly, genetic removal of these cells aggravated perivascular fibrosis and worsened cardiac function, suggesting a protective role for premature senescent cardiac myofibroblasts.⁶⁶ In contrast, ischaemia reperfusion injury causes premature senescence in both cardiac myocytes and fibroblasts. Clearance of these cells by a senolytic drug improved cardiac function and reduced cardiac fibrosis.⁶⁷

4. Pathological fibroblast fate in blood vessels

4.1 Perivascular fibroblasts with ang II infusion

The arterial wall is a heterogeneous three-layered structure composed of an inner membrane, tunica media, and outer membrane. The outer membrane stroma consists of ECM scaffold containing fibroblasts, immune cells (dendritic cells and macrophages), progenitor cells, vasa vasorum endothelial cells, pericytes, and adrenergic nerves, making the outer membrane the most complex and heterogeneous compartment of the vessel wall. Fibroblasts make up the majority of the cells of the outer membrane and are responsible for the deposition of abundant collagen fibres around blood vessels.⁶⁸ Compared with cardiac fibroblasts, fewer studies have focused on the origin of perivascular fibroblasts in pathological adventitial fibrosis. Genetic tools to track adventitial cell types and their potential contribution to vascular fibrosis were reviewed recently.¹⁴

A mouse line with S100A4 promoter Cre mice has been used to delete Atgra1 (gene encoding the mouse major Ang II AT₁ receptor, AT_1A) in fibroblasts. In the S100A4-Cre AT_1A receptor-deleted mice, Ang II infusion-induced hypertension was unaltered. However, Ang II-induced medial thickening and increase in cell numbers were reduced in the ascending but not descending aorta in the fibroblast AT_1A receptor-deleted mice. In ascending aortas, S100A4-promoter driven LacZ positive cells were increased in adventitia as well as in medial layers upon Ang II infusion whereas there was no change in LacZ positive cell numbers in descending aortas.⁶⁹ These results indicate that S100A4 ECM-Fib in the ascending aorta has a unique role in hyperplastic response in mice with chronic Ang II infusion.

Studies utilizing inducible Col1a2-Cre mice demonstrated that vascular mesenchymal cells mediate adventitial fibrosis and immune cell infiltration via a nuclear factor κ-B/RelA-dependent mechanism in mice with Ang II infusion.⁷⁰  Col1a2-Cre mediated deletion of Nox2⁷¹ or G protein-coupled receptor kinase 5⁷² also attenuated vascular thickening or perivascular fibrosis induced by Ang II. However, Col1a2-Cre reporter can be expressed in both adventitial fibroblasts and VSMCs.⁷³ Signalling mechanisms and updated roles of AT₁ receptors in hypertensive cardiovascular remodelling including fibrosis were reviewed recently.^74,75

4.2 Perivascular mesenchymal stem cells in Ang II and TAC models

MSCs reside in the perivascular niche of many organs. Sca1 and CD34 have been used to identify MSCs that can be differentiated into VSMCs or endothelial cells (reviewed in¹⁴). Recently, Gli family zinc finger 1 (Gli1) which mediates sonic hedgehog signalling, was reported as a novel MSC marker.¹⁴ Genetically labelled Gli1-MSC positive cells were found in perivascular niche in many organs including small vessels in heart and kidney as well as aortas, and consistently expressed the mesenchymal marker PDGFRβ.⁴³ Upon Ang II infusion for 4 weeks, Gli1 positive cells expanded to coronary artery perivascular fibrosis area and co-stained with αSMA suggesting that Gli1 positive cells were differentiated into myofibroblasts. Gli1/αSMA positive cells were also induced at cardiac perivascular and interstitial fibrosis area at 8 weeks after TAC injury.⁴³  Gli1/αSMA positive cells accounted for 60 and 63% of total αSMA positive myofibroblasts induced by Ang II infusion or TAC, respectively. In addition, ablation of Gli1 positive cells reduced cardiac fibrosis, hypertrophy, and preserved cardiac function in mice upon TAC injury.⁴³ BM transplant experiments further suggest that Gli1 positive residential MSCs are a primary source of myofibroblasts contributing to tissue fibrosis.⁴³ However, the expression of periostin was not investigated in this study.

In contrast, recent studies using mice with Ang II infusion suggest that the main sources of activated adventitial fibroblasts are differentiated from either BM derived Sca1 positive progenitor cells⁴⁷ or VSMC-derived Sca1 positive progenitor cells.⁷⁶ The BM derived Sca1 and CD45 positive cells represent major collagen producing cells and are increased in aorta upon Ang II infusion for 2 weeks.⁴⁷ While myofibroblasts and periostin expression were not defined in this manuscript, these Sca1-positive cells likely represent activated fibrocytes. Using mature SMC fate mapping via inducible Myh11-Cre tracer mice and constitutive SM22α-Cre tracer mice, the ability of mature SMC in the vascular media to migrate and differentiate to adventitia media boarder αSMA negative Sca1/CD34 positive progenitor cells (termed AdvSca1 cells) has been demonstrated.⁷⁷ Following carotid artery ligation injury for 3 days, the number of AdvSca1 cells in adventitia was increased. In addition, mature SMC-derived cells differentiated and consisted minor population of CD45-positive cells consistent with the concept that resident SMC derived progenitor cells are a major source of fibrotic cells in response to arterial injury.⁷⁷ Subsequent study on this concept demonstrated that the VSMC-derived [SM22α-yellow fluorescent protein (YFP) positive] AdvSca1 progenitor cells represent Gli1 positive collagen-producing myofibroblasts and were increased in adventitia upon carotid artery ligation injury.⁷⁶ Under a baseline condition, compared with non-SMC AdvSca1 cells, SMC AdvSca1 cells preferentially express genes associated with hedgehog/Wnt/β-catenin signalling, ECM and ECM-modifying, including Gli1, Gli2, Col1a1, and Lox. While not discussed in this manuscript SMC AdvSca1 cells also preferentially express bone morphogenetic protein 1 coding Bmp1, Bmp2, and Comp suggesting the potential to be matrifibrocyte progenitor cells. In response to carotid artery ligation injury, SMC AdvSca1 cells showed down-regulation of stemness-associated genes (Ly6a/Sca1, Cd34, Klf4) and hedgehog/Wnt signalling (Gli1, Gli2, Wnt2b) and up-regulation of genes associated with an activated fibroblast phenotype (Postn, Tgfb1, Fn1), macrophage phenotype (Cd68, Lgals3/galectin 3, Adgre1/adhesion G protein-coupled receptor E1), and inflammatory factors (Il1b, Ccl2, Tnf, Nfkb2). To distinguish mature SMC and SMC AdvSca1 cells, inducible Gli1 reporter mice were utilized. Gli1 traced AdvSca1 cells expand at adventitia but not at intima in response to vascular injury. These cells were mostly Postn positive, CD68 negative, and had lost Sca1 expression.⁷⁶ Flow cytometry was used to determine if injured Gli1 cells express αSMA. While αSMA positive cells doubled upon injury, Gli1 cells expressing αSMA were only seen at media but not at adventitia (myofibroblasts) 3 days, 7 days, and 3 weeks post-injury. In addition, Gli1-Cre mediated deletion of Klf4 (Krüppel-like factor 4) in AdvSca1 cells led to spontaneous adventitial remodelling suggesting that stemness-associated genes are important to harness dedifferentiation of AdvSca1 cells to activated fibroblasts. An increase in Gli1 positive population in aortic adventitia was also confirmed in mice with Ang II infusion for 4 weeks.⁷⁶

TAC model was recently used to trace Pdgfra positive fibroblasts from MSC lineage cells in mice. At day 28 post-TAC, ascending aortas showed adventitial hyperplasia. These adventitial cells were PDGFRα positive, mildly αSMA positive (compared to strongly positive medial VSMCs) but negative for VSMC marker SMMHC/Myr11.⁷⁸ PDGFRα positive fibroblasts close to the boundary of the media were also strongly positive for Sca1 (other adventitia area was mildly positive for Sca1) and CD34. Increase in Pdgfra-Sca1 positive cells by ligation and wire injury at adventitia⁷⁸ also suggests induction of AdvSca1 cells in this study. Immune cell markers were not tested in these experiments.⁷⁸ This study confirms the aforementioned AdvSca1 SMC concept that the major source of activated fibroblasts may be mature SMC derived adventitial Sca1/Gli1 positive progenitor cells. However, follow up studies are desired to further explore the relationship of these Sca1 positive SMC/Gli1 lineage-activated fibroblasts in hypertensive vascular fibrosis and hypertension. Table 3 summarizes perivascular fibroblast lineage tracing studies in mouse models of neurohormonal and mechanical stress. Figure 3 illustrates our updated understanding of the fibroblast cell types involved in perivascular fibrosis induced by mechanical (TAC and ligation) stress or neurohormonal (Ang II) stress. Two major subtypes are matrifibrocytes and fibrocytes are also important cell types for cardiac interstitial fibrosis. The major collagen producing matrifibrocytes seen in vascular adventitia may be similar to Postn-expressing ECM-Fb and Thbs4-Fb found in the cardiac fibroblast populations. Fibrocytes may have similar roles to interferon fibroblasts seen in the heart upon TAC or Ang II infusion. While induction of myofibroblasts by Ang II infusion was observed in the vasculature, myofibroblast contribution to perivascular fibrosis seems limited.

Figure 3

Current understanding of fibroblast cell types involved in perivascular fibrosis induced by mechanical and neurohormonal stress. Matrifibrocytes and fibrocytes are two major subtypes identified as contributing to perivascular fibrosis and vascular inflammation. The major collagen producing matrifibrocytes seen in vascular adventitia may be similar to those found in the cardiac fibroblast populations. Resident MSC is the source of these activated fibroblasts. The perivascular MSC may have mature VSMC and BM cell lineage. Fibrocytes may have similar roles as interferon fibroblasts seen in the heart upon TAC or Ang II infusion. Again, the contribution of myofibroblasts to perivascular fibrosis seems relatively limited. Abbreviations: αSMA, smooth muscle α actin; AdvSca1, adventitial Sca1; Ang II, angiotensin II; BM, bone marrow; Fib, fibroblast; MSC, mesenchymal stem cell; Sca1, stem cell antigen 1; TAC, Transverse aortic constriction; VSMC, vascular smooth muscle cell. Italics indicate the marker genes.

Table 3

Perivascular fibroblast lineage tracing under hormonal and mechanical stress

References	Model	Marker	Quiescent	Activated Fib	Matrifibrocyte	Matrifibrocyte
Lu76	Ang II	Gli1-CD68	All CD68 –			+++/–a
Wu47	Ang II	Sca1-Col1	Low		High
Lu76	Ligation		Control	Day 3	Day 7	Day 21
		Gli1-αSMA	Low	30∼40%b	30∼40%	30∼40%
		Gli-Postn	Low		++	+
		Gli1-EdU	–	+++	++	+
Kimura78	Ligation		Control		Day 28	Day 56
		Pdgfa-Sca1			High at adventitia
		Pdgfa-Elastin				High at neointima
Kimura78	Wire			Day 14
		Pdgfa-Sca1		+ at adventitia
		Pdgfa-αSMA		++ at neointima
Kimura78	TAC		Control		Day 28
		Pdgfa-Sca1	Low		High at adventitia
		Pdgfa-αSMA	Low		Mild at adventitia

Italics indicate the marker genes.

αSMA, smooth muscle α actin; Ang II, angiotensin II; col1, collagen 1; EdU, 5-ethynyl-2′-deoxyuridine; Sca1, stem cell antigen 1; TAC, Transverse aortic constriction.

^aBoth CD68 positive and negative Gli1 cells increased at adventitia.

^b% of total Gli1 positive (A marker for adventitial Sca1 positive smooth muscle progenitor cells).

5. Perivascular cell single-cell transcriptomics

5.1 Hypertension and Ang II models

scRNA-seq was performed in aorta and mesenteric artery from normotensive Wister Kyoto rats (WKY) and spontaneous hypertensive rats (SHR). Mesenchymal stromal cell population from SHR significantly expressed osteo-inductive Bmp2, osteogenic marker Alpl, hypertension associated Col8a1, Tgfb1 and Cilp. Thus, matrifibrocytes may be enriched in SHR arteries. Ten subclusters were observed in arterial mesenchymal stromal cells. Cluster 7 preferentially expresses Acta2, SM22α-coding Tagln, and Postn, thus representing the activated myofibroblast cluster. This cluster consisted less than 10% of the mesenchymal stromal cells in all arteries showed slight increase and decrease in SHR aorta and mesenteric arteries, respectively. Changes in cluster populations showed some common and unique regulation among large and small arteries in SHR compared with WKY including common and aorta-specific increases in clusters defined by expression of Col8a1 and oxidative and endoplasmic reticulum (ER) stress-associated thioredoxin interacting protein-coding Txnip,⁷⁹ respectively.

scRNA-seq was also performed in a mouse model of abdominal aortic aneurysm (AAA) with Ang II-infused apolipoprotein E-/- mice. Three fibroblast clusters (all expressing Cd34 and Pdgfra) were identified, of which only the high Acta2 low Cd34 myofibroblast cluster cells were increased in AAA aortas.⁸⁰ Interestingly, separate from these fibroblast clusters, a macrophage cluster (Cd68 positive CD45 encoding Ptprc positive) population was significantly increased in AAA. Within the macrophage cluster, a significant increase in the fibrocyte population (Cd34/Col1a2 positive) was observed. Cd45/Col1a2 positive cells covered >95% of the population with significant expression of collagens and other ECM-related genes including Postn.⁸⁰ These cell types may be similar to the Sca1 lineage fibrocytes seen in mouse aorta upon Ang II infusion.⁴⁷

Recently, lineage tracing data from Myh11-Cre mice was combined with scRNA-seq analysis in an Ang II infusion model of aortic aneurysm and dissection. Ang II (1000 ng/kg/min) was infused for 7 days and the outcomes were classified and analyzed for ascending aorta dilatation/aneurysm and ascending aorta dissection compared to control mice with saline infusion.⁸¹ Among the VSMC clusters, a slight but significant decline in contractile cluster population was observed (control ∼32%, dilatation ∼28%, and dissection ∼23%). In contrast, ECM regulating VSMC cluster was increased (12 to ∼15%) in AngII infusion groups. Interestingly, increase in contractile/inflammatory VSMC cluster was only observed with dissection. Other notable findings in this study are that Myh11-traced VSMC lineage cells were also found in fibroblast cluster and macrophage cluster, confirming that mature SMC differentiated to activated fibroblast and macrophage. The SMC lineage macrophages preferentially expressed genes involved in ECM organization, adhesion, contraction, and wound healing, thus representing activated fibrocytes. While the fibrocyte population increased upon Ang II infusion in ascending aortas, the SMC lineage fibroblast population declined with Ang II infusion.⁸¹ Thus, induction of SMC-lineage adventitial ECM producing cells upon Ang II infusion in aorta as suggested⁴⁷ was not confirmed in this study. Incidence of dilatation in C57Bl6 mice at day 7 of Ang II infusion was limited whereas microdissection was seen in more than 90% of aortas.^82,83 Thus, these findings could be specific to the aortic phenotypes.

5.2 Atherosclerosis and aging models

In addition to the luminal side mechanism, alterations in the adventitial wall including inflammatory fibroblast activation have been implicated in the development of atherosclerosis.⁸⁴ scRNA-seq in aortic adventitial cells from apolipoprotein E-/- mice demonstrated Sca1 positive mesenchymal cell clusters are pro-inflammatory with increased CCL2 secretion.⁸⁵ In addition, while involvement in vascular fibrosis was unclear, upon femoral artery wire injury, Sca1/PDGFRα positive fibroblasts (Gli1-AdvSca1 cells?) migrated to media and differentiated to regenerative VSMC.⁸⁶

Aging aorta showed enhanced stiffness which is associated with adventitial fibrosis. However, the role of fibroblast activation in this process remains mostly unclear.⁸⁷ Recently, scRNA-seq was performed in young (12 weeks) and aged (72 weeks) mouse aortas.⁸⁸ Upon removal of immune cells and endothelial cells, PDGFRα and Dipeptidase 1 were found to be fibroblast selective markers (not expressed in VSMCs). In the aortic adventitial fibroblasts, 12 different clusters were identified with three trajectories demonstrating three representative markers (complement activating CD55, immune activating chemokine Cxcl14, and ECM cross-linking LOX). Compared with the young control mice, aged, or low density lipoprotein (LDL) receptor -/- mice showed increase in adventitial collagen deposition. Aged mouse aortas have more CD55 and CXCL14 positive fibroblasts, whereas atherosclerotic LDL receptor -/- aortas were enriched with CXCL14 and LOX positive fibroblasts. Since all aortas did not show overt pathology as well as inflammatory cell infiltration, the phenotype changes of fibroblasts precede adventitial remodelling in these models. In advanced atherosclerotic aortas, only CD55 positive fibroblasts were increased, and these cells were highly positive for the stem cell marker Sca1.⁸⁸ Enhancement of Sca1 positive cells is consistent with a prior report showing enrichment of Sca1 positive pro-inflammatory fibroblasts in hyperlipidemic apolipoprotein E-/- aortas.⁸⁴

6. Perivascular fibroblasts as origin of myofibroblasts in fibrotic kidney disease

In previous studies, Ang II infusion in mice was shown to increase CD45/collagen I double-positive cells as well as αSMA positive cells in the kidney suggesting that renal myofibroblasts and fibrocytes were induced in Ang II model of hypertension.⁸⁹ The aforementioned Gli1 and αSMA positive myofibroblasts were seen in injured kidney in response to ischaemia reperfusion and unilateral ureteral obstruction (UUO).⁴³ Recently accumulating evidence indicates that pericytes and perivascular fibroblasts are the origins of myofibroblasts in fibrotic kidney disease.^90,91 Proximal tubule-specific tracing mice were used to exclude epithelial transition to αSMA positive myofibroblast in kidney fibrosis in response to UUO.⁹² Parabiosis experiments further demonstrated that only a small fraction of αSMA positive myofibroblasts derive from circulating CD45 positive cells. These fibrocytes were also positive for PDGFRβ, a mesenchymal marker. scRNA-seq confirms these findings and further suggests a paracrine contribution of circulating immune cell-derived fibrocytes to renal fibrosis.⁹² Findings from human kidney cells support these experimental findings. Kidney cell samples from hypertensive nephrosclerosis and control samples were analyzed for scRNA-seq.⁹³ Activated ECM-producing fibroblasts (termed myofibroblasts in the manuscript regardless of the expression of Acta2) seen in hypertensive human kidneys are strongly positive for periostin and derived from resident fibroblasts and pericytes. PDGFRα and PDGFRβ also marked these activated fibroblasts.⁹³ PDGFRα and PDGFRβ were found to be better markers than αSMA for characterizing the activated kidney fibroblasts induced by ischaemic injury when analyzed by scRNA-seq.⁹⁴ Periostin seems to be a common marker detecting ECM-producing fibroblasts in hypertensive fibrotic organs including heart and kidney.

7. Mechanisms of ECM-producing fibroblast induction in hypertension

7.1 Intracellular mechanisms

Several mechanisms have been reported for how pressure overload converts fibroblasts into myofibroblasts (or ECM-producing fibroblasts) thus promoting cardiac fibrosis.⁹⁵ A prior review article summarized the classical understanding of myofibroblast induction utilizing myofibroblast regulated gene markers to track potential signalling mechanisms.⁹⁵ Hormonal extracellular factors and mechanical stress on the resident fibroblasts are the two major mechanisms for myofibroblast induction. The main extracellular factors include TGF-β, Ang II, and endothelin-1. Mechanical stress acts directly via mechanosensors including transient receptor potential cation channel subfamily C member 6/TRPC6. However, many of the referenced studies were limited to cell culture system and or relied exclusively on αSMA for activated fibroblast detection, with a few exceptions. Careful interpretation seems needed in cell culture data dealing with fibroblasts and myofibroblasts. This is because quiescent fibroblasts are prone to rapidly differentiate from myofibroblasts in a cultured condition.⁹⁶ Methods for isolation and culture of adult murine cardiac fibroblasts and myofibroblasts have been described. Seeding on compliant substrates (elastic modulus 5∼8 kPa) maintains αSMA negative quiescent fibroblasts.^97,98

As mentioned above, TGF-β likely promotes myofibroblast induction and activation via TGF-β receptor 1/2 and Smad3 signalling.³⁶ It has been proposed that fibrocytes are responsible for Ang II mediated cardiac collagen production. MCP-1 was identified as a factor secreted by fibrocytes that mediate activated fibroblast phenotype including TGF-β induction.⁴⁵ In addition, enhanced glycolysis seems to be a common cellular mechanism for transition from quiescent fibroblasts to myofibroblasts in response to TGF-β or Ang II.⁹⁹

p38 stabilizes yes-associated protein (YAP) the transcriptional coactivator in the Hippo pathway, which is negatively regulated by the kinases, large tumour suppressor kinases (Lats), Lats1, and Lats2. Thus Lats1/2 knockout mice had increased baseline and post MI fibrosis via induction of myofibroblasts.¹⁰⁰ Fibroblast-selective deletion of YAP decreased fibrosis upon MI or Ang II/PE infusion.¹⁰¹ Several YAP and p38 inhibitors were shown to be effective in treating pathological fibrosis in animal models including MI and Ang II infusion.¹⁰²

Recently, progress was made regarding the signalling mechanism of fibroblast differentiation and activation in mouse models of hypertension using Postn gene-targeted mice. Zempo et al.¹⁰³ examined Postn-positive cell-specific deletion of KLF5, a transcriptional mediator of cardiovascular remodelling in mice and showed that KLF5 is involved in the induction of myofibroblasts in hypertension. KLF5 appears essential for migration of αSMA/FSP1 double-positive myofibroblasts to the aortic tunica media associated with hypertension. Shimizu et al.¹⁰⁴ used Postn-specific deletion of Rho-associated coiled-coil containing kinase 2 (ROCK2), a key regulator of integrin activation and cytoskeletal dynamics, to show that ROCK2 is required for cardiac fibrosis and hypertrophy via induction of connective tissue growth factor/CTGF and fibroblast growth factor 2/FGF2.

7.2 Extracellular mechanisms

Extracellular mechanisms such as communication with immune cells are additionally critical for induction of myofibroblasts. T cell KLF10 deletion augmented Ang II-induced perivascular but not interstitial fibrosis in the heart and kidney. KLF10 appears to suppress T cell interleukin-9 production which promotes fibroblast ECM production.¹⁰⁵ It is also interesting to note that inducible (SMMHC-Cre) as well as constitutive (SM22α Cre) silencing of smooth muscle AT_1A receptor markedly attenuated perivascular fibrosis in mice with Ang II infusion.¹⁰⁶ The SM22α Cre-mediated deletion of smooth muscle ADAM17 also attenuated Ang II-induced perivascular fibrosis but had no effect on hypertension.¹⁰⁷ These data suggest that VSMC signalling has a paracrine role in hypertensive fibrosis. However, since SMMHC and or SM22α driver mice were utilized in these studies, one cannot exclude the potential contribution of AT_1A receptor and subsequent ADAM17 activation in SMC-derived AdvSca1 cells to hypertensive perivascular fibrosis. Paracrine TGF-β receptor II signalling also seems to be an important inducer of cardiac myofibroblasts and fibrosis under proteotoxic stress seen with crystallin AB mutation. Mouse hearts with crystallin AB R120G mutation cause cardiac fibrosis with induction of myofibroblast markers and activation of the TGF-β cascade.¹⁰⁸  Postn-Cre mediated deletion of TGF-β receptor II reduced fibrosis and cardiac hypertrophy in the mutant mice.

For the fibroblast transition to myofibroblast in response to pressure overload, T cells may play a significant role. The data suggest that upon TAC, Th1 effector cells drive cardiac fibrosis via secretion of interferon-γ, which promotes fibroblast secretion of TGF-β and subsequent transition to myofibroblasts,¹⁰⁹ although this study was performed mostly with in vitro experiments. T cells are activated through interaction with antigen-presenting cells such as dendric cells and macrophages that have processed antigens and uploaded them into cell surface major histocompatibility complex II (MHCII). The MHCII loaded with the antigen is then recognized by the T cell receptor to activate Th1 cells for interferon-γ production. It was shown that cardiac fibroblasts were able to express antigen-loaded MHCII and conditional deletion of fibroblast MHCII using Tcf21-Cre mice attenuated perivascular fibrosis and improved cardiac function in the TAC model.¹¹⁰

8. Removal of activated fibroblasts as a potential treatment for cardiovascular fibrosis

Based on the knowledge that removal of activated fibroblasts/myofibroblasts may be beneficial against hypertensive or post MI cardiac fibrosis, chimeric antigen receptor (CAR) T cell therapy was designed to target cardiac fibrosis in mice. In the first study, Postn-Cre mice were used to express xenogenic antigen on the surface of activated fibroblasts in the hearts. The mice were infused with Ang II plus PE to induce cardiac fibrosis. Adoptive transfer of CD8 positive T cells that express the antigen receptor significantly attenuated cardiac interstitial fibrosis.¹¹¹ In this study FAP mRNA encoding a cell-surface glycoprotein was identified as significantly over-expressed in human hypertrophic cardiomyopathy or dilated cardiomyopathy.¹¹¹ Cardiac fibroblast FAP expression was confirmed with MI model, TAC, and Ang II plus PE. Adoptive transfer of FAP CAR T cells a week after Ang II plus PE infusion removed activated fibroblasts, attenuated cardiac fibrosis, and improved cardiac function.¹¹¹ This group subsequently tested T cell targeting lipid nanoparticles loaded with modified mRNA encoding a CAR designed against FAP. The lipid nanoparticle attenuated cardiac fibrosis and improved cardiac function in Ang II plus PE infusion model.¹¹² Since such technology was already successful against COVID-19, this study demonstrated strong translational value of the CAR T technology against cardiac fibrosis.

9. Perspectives

Recent advancements in omics experiments and in vivo lineage tracing significantly advanced our knowledge of myofibroblast origin and markers seen in cardiovascular diseases. αSMA may not be an ideal marker for activated ECM-producing fibroblasts (ECM-fib/matrifibrocytes), contributing to fibrosis seen in cardiovascular disease including hypertension. Using a combination of two or three markers is ideal, but they should be selected in each context (myofibroblast, matrifibrocyte vs. fibrocyte, hypertension/TAC vs. MI, reactive vs. replacement fibrosis, as well as interstitial vs. adventitial fibrosis). Intracellular signalling mechanisms of activated fibroblast induction and characteristics of relevant fibroblast subpopulations become clearer by using novel techniques (Fate mapping and scRNA-seq), whereas those with hypertension may further involve extracellular fibroblast communication such as with VSMC and immune cells. A few studies suggest that targeting mechanisms of active fibroblast induction in hypertension may be a promising add-on therapy for treatment of human hypertension. However, our knowledge of these mechanisms in hypertension is still limited. It is important to test potential mechanisms in several animal models of hypertension. Additionally, these experimental findings need to be corroborated in studies of human hypertension.

Supplementary material

Supplementary material is available at Cardiovascular Research online.

Data availability

No new data were generated or analyzed in support of this research.

Author notes

Conflict of interest: None declared.