Human ESC-derived vascular cells promote vascular regeneration in a HIF-1α dependent manner

Abstract Hypoxia-inducible factor (HIF-1α), a core transcription factor responding to changes in cellular oxygen levels, is closely associated with a wide range of physiological and pathological conditions. However, its differential impacts on vascular cell types and molecular programs modulating human vascular homeostasis and regeneration remain largely elusive. Here, we applied CRISPR/Cas9-mediated gene editing of human embryonic stem cells and directed differentiation to generate HIF-1α-deficient human vascular cells including vascular endothelial cells, vascular smooth muscle cells, and mesenchymal stem cells (MSCs), as a platform for discovering cell type-specific hypoxia-induced response mechanisms. Through comparative molecular profiling across cell types under normoxic and hypoxic conditions, we provide insight into the indispensable role of HIF-1α in the promotion of ischemic vascular regeneration. We found human MSCs to be the vascular cell type most susceptible to HIF-1α deficiency, and that transcriptional inactivation of ANKZF1, an effector of HIF-1α, impaired pro-angiogenic processes. Altogether, our findings deepen the understanding of HIF-1α in human angiogenesis and support further explorations of novel therapeutic strategies of vascular regeneration against ischemic damage.


Introduction
Ischemic conditions are characterized by reduced blood flow and consequent insufficient oxygen and nutrient supply, causing severe tissue injury (Lei et al., 2021;Van Nguyen et al., 2021).If not resolved quickly, low levels of intracellular ATP and acidic pH levels trigger exacerbated calcium influx in plasma and mitochondria, ultimately causing cell death (Kalogeris et al., 2012).To ameliorate the harsh microenvironment of low oxygen supply and nutrient deprivation caused by ischemia, promotion of angiogenesis to restore the blood flow is considered a promising therapeutic approach (Wahlberg, 2003;Vrselja et al., 2019;Wang and Qin, 2023).However, current clinical therapies such as thrombolytic or vasodilator drugs and surgery fall significantly short of promoting angiogenesis and vascular remodeling efficiently (Bian et al., 2019), and therefore, there is a vast need to investigate mechanistic underpinnings of vascular regeneration for directing development of effective treatments.
Blood vessel mainly consists of three layers including the innermost tunica intima, the middle tunica media, and the outermost tunica adventitia, which are primarily composed of three cell types: vascular endothelial cells (VECs), vascular smooth muscle cells (VSMCs), and mesenchymal stem cells (MSCs) (Wang et al., 2018;Ling et al., 2019;Yan et al., 2019).In particular, MSCs located in the adventitia layer and commonly referred to as vascular wall-resident MSCs are critical for local capacity of neovascularization in disease processes (Ergun et al., 2011;Worsdorfer et al., 2017).Vascular cell activation and endogenous angiogenesis are essential to recover the oxygen supply and boost the repair of the ischemia-induced injured tissues.Several angiogenic growth factors such as VEGF, PDGF, and FGF2 are known to be upregulated upon ischemic insult and act on the corresponding receptors in vascular beds, consequently inducing sprouting and capillary growth toward the ischemic tissue (Dor and Keshet, 1997;Vimalraj, 2022).However, the molecular mechanisms intrinsic to the human vascular cell types underlying ischemic vascular remodeling remain largely unexplored.
Hypoxia-inducible factor (HIF-1α) is a central transcription factor that detects cellular oxygen levels and rapidly responds pathophysiological ischemia.Different from its dimerized partner, constitutively expressed β-subunit (HIF-1β), HIF-1α is sensitive to changes in oxygen levels.Under normoxia, HIF-1α proteins are rapidly hydroxylated by prolyl hydroxylase domain enzymes and degraded.However, hypoxia inhibits the hydroxylation of HIF-1α, preventing its degradation and leading to its accumulation and translocation into the nucleus (Maxwell et al., 1999;Ivan et al., 2001;Jaakkola et al., 2001).In the nucleus, HIF-1α promotes angiogenesis by transcriptionally activating the expression of canonical pro-angiogenic factors, including VEGF, PLGF, PDGFB, and ANGPT1, and pro-angiogenic chemokines and receptors, such as SDF-1, S1P, CXCR4, and S1PR (Zimna and Kurpisz, 2015;Cai et al., 2022;Feng et al., 2022;Vimalraj, 2022).However, clinical trials have demonstrated that supplementation of these angiogenic factors is usually insufficient to relieve ischemic diseases (Annex and Cooke, 2021).Most importantly, how HIF-1α regulates the physiological functions of different human vascular cells and what the downstream genes of HIF-1α intrinsic to vascular cells are, remain enigmatic.Consequently, gaining insights into such mechanisms are of great importance for developing new therapeutic approaches for ischemic damage and associated diseases.
In this study, we used CRISPR/Cas9-mediated gene editing to generate HIF-1α-deficient human embryonic stem cells (hESCs) and subsequently differentiated these into VECs, VSMCs, and MSCs, the three major vascular cell types.Our data uncovered that human ESC-derived vascular cells promote ischemic vascular regeneration and rescue ischemic damage in a HIF-1α dependent manner.Strikingly, MSCs exhibited the highest susceptibility to HIF-1α deficiency.Through molecular profiling across the vascular cell types, we identified ANKZF1 as a major effector gene downstream of HIF-1α in mediating angiogenesis in MSCs.Overall, this study identifies novel therapeutic targets for development of approaches to promote vascular regeneration and counteract ischemic diseases.

Protein & Cell
HIF-1α deficiency does not influence the differentiation capabilities towards human vascular cells.
We subsequently sought to explore in which way HIF-1α contributes to the angiogenic potential of human vascular cells.It is well known that cell migration and in vitro formation of capillary-like tubes are crucial for angiogenesis (Zhang et al., 2020b;Ghaffari-Makhmalbaf et al., 2021;Wang et al., 2022a).First, we examined cell migration capability and observed enhanced cellular migration in wild-type (WT) hVECs, hVSMCs, and hMSCs in response to hypoxia compared to normoxic conditions (Fig. 2L).As expected, cellular migration upon induction of hypoxia was compromised in all three types of human HIF-1α-ablated vascular cells (Fig. 2L).And, consistent with the notion that hypoxic condition boots angiogenesis (Pugh and Ratcliffe, 2003), we noticed an increment in the cumulated tube length in WT human vascular cells upon exposure to hypoxia (Fig. 2M).However, the tube formation in HIF-1α-deficient human vascular cells was impaired relative to their WT counterparts, as evidenced by the diminished cumulated tube lengths (Fig. 2M).Overall, these findings elucidated that the HIF-1α signaling cascade is indispensable for hypoxia-induced human vascular cell activation and angiogenis.

Vascular remodeling and repair upon ischemia damage are compromised in HIF-1α-deficient human vascular cells
Next, to inspect the function of HIF-1α in human vascular cells on neovascularization in vivo, we used a well-established murine model of hindlimb ischemia with femoral artery ligation (Yang et al., 2017;Yan et al., 2019).First, we performed the laser doppler perfusion monitoring assay to measure the local microcirculatory blood perfusion after surgery (Fig. 3A).Compared with hindlimbs without femoral artery ligation, block of blood flow was noticed in the surgery group (Fig. 3B).Intriguingly, when we measured the local microcirculatory blood at different time points after cell implantation, we found that implantation of a mixture of HIF-1α +/+ hVECs and hVSMCs into the ischemic legs led to a more rapid recovery of blood flow compared to those implanted with HIF-1α −/− cells (Fig. 3B).These observations suggest that HIF-1α deficiency impairs the angiogenesis-promoting beneficial effect of human vascular cells.In accordance with the aforementioned observations, capillary density, as indicated by CD31-positive cells, was also remarkably increased by implantation of HIF-1α +/+ cells relative to HIF-1α −/− cells at the tissue level (Fig. 3C).Concurrently, we observed that ischemia-induced increase in fibrosis, a hallmark feature of ischemic damage (Stabile et al., 2003;Zhang et al., 2022a), was diminished upon HIF-1α +/+ cell implantation relative to what we observed in the HIF-1α −/− cell delivery group (Fig. 3D).Since it is well known that ischemia induces inflame ischemic lesions (Eltzschig and Carmeliet, 2011), we next examined inflammation levels of ischemic tissues with or without human vascular cell transplantation.Immunofluorescence staining of CD45, a pan-marker for immune cells (Altin and Sloan, 1997;Geng et al., 2022), showed that the infiltration of CD-45 positive immune cells in the hindlimb was markedly alleviated by delivery of a mixture of HIF-1α +/+ hVECs and hVSMCs to local lesions relative to delivery of HIF-1α −/− counterparts (Fig. 3E).More strikingly, TNF-α positive area was also less in the HIF-1α +/+ cell-implanted group compared to that in HIF-1α −/− group (Fig. 3F).Collectively, these data indicated that HIF-1α ablation compromises the pro-angiogenic role of human vascular cell function under ischemic condition.
It is well accepted that activation of MSC-like cells in adventitial wall also play a critical role on vascular protection and regeneration (Vono et al., 2012).Indeed, when implanted into ischemic hindlimb, we found that HIF-1α +/+ hMSCs but not HIF-1α −/− hMSCs induced a superior recovery of blood perfusion in the hindlimb ischemia mouse model (Fig. 3G and 3H).Consistently, capillary density, as assessed by quantification of CD31positive cells in hindlimb muscles, was only increased after transplantation of HIF-1α +/+ hMSCs compared to the Vehicle control (Fig. 3I).Moreover, ischemia-induced limb fibrosis was ameliorated by HIF-1α +/+ hMSC alone (Fig. 3J).In addition, HIF-1α +/+ hMSCs also attenuated the ischemia-induced inflammation characterized by massive infiltration of CD45-positive immune cells and elevated release of inflammatory cytokine TNF-α, which was not the case upon transplantation of HIF-1α −/− hMSCs (Fig. 3K and 3L).Altogether, these data indicated that hMSC transplantation both augments angiogenesis and blunts fibrosis and inflammation after ischemia, while knockout of HIF-1α abrogates the angiogenic and therapeutic potential of hMSCs.
Next, we focused on analyzing overlapping genes between upregulated DEGs by hypoxia exposure in WT cells and downregulated ones in HIF-1α −/− vs. HIF-1α +/+ cells after hypoxia exposure, which we referred to as hypoxia-induced HIF-1α responsive genes (HHRGs) (Fig. 4B).As shown by Venn diagram, hMSCs contained the most HHRGs (54 genes in hVECs, 40 genes in hVSMCs, and 372 genes in hMSCs, respectively) (Fig. 4B), which was concordant with the highest transcriptional fluctuations observed in hMSCs (Fig. 4A).Through Gene Ontology (GO) term and pathway enrichment analysis, we discovered that although these HHRGs were divergent across three cell types (Fig. 4B and 4D), they functionally converged on "blood vessel development" (e.g., ANGPT2 and ANGPTL4 in hMSCs, FAP and PROK1 in hVSMCs, LOXL1 and THBS1 in hVECs) and "response to hypoxia" (e.g., AK4, HK2, PDK1, and SLC2A1 in hVECs and hMSCs, PLOD2 and PRKCE in hVECs) (Figs.4C, 4D and S1F).Notably, three classical HIF-1α target genes (SLC16A3, CTHRC1, and LDHA) were shared across all three cell types (Fig. 4E and 4F).Among these, SLC16A3 encodes a member of the solute carrier family-16, which catalyzes lactic acid and pyruvate transport across the plasma membranes (Contreras-Baeza et al., 2019); CTHRC1 encodes a secretory protein, collagen triple helix repeat containing 1, which is involved in the cellular response to arterial injury through facilitation of vascular remodeling (Pyagay et al., 2005).Moreover, amongst the top 10-ranked HHRGs of different vascular cells, some were shared across three cell types and some exhibited cell type specificity (Fig. 4G).For example, LDHA, encoding lactate dehydrogenase A that catalyzes the conversion of L-lactate and nicotinamide adenine dinucleotide to pyruvate and hydrogenated nicotinamide adenine dinucleotide, was shared by three cell types, which suggested its central role in anaerobic glycolysis closely relevant to vessel sprouting (De Bock et al., 2013;Valvona et al., 2016;Du et al., 2021).VWF, specific to hVECs, encodes a glycoprotein responsible for hemostasis by promoting adhesion of platelets to the sites of vascular injury (Chen and Lopez, 2005).Overall, our data revealed that HIF-1α transcriptionally activated different sets of downstream genes under hypoxia condition, which may convergently mediate pro-angiogenic functions in human vascular cells.

ANKZF1 acts as an effector gene downstream of HIF-1α in hMSCs
Given the highest susceptibility of hMSCs manifested by altered transcriptomic profiling, we next explored the mechanism underpinning HIF-1α-mediated pro-angiogenic capacity in hMSCs.Through a conjoint analysis of HHRGs containing the canonical HIF-1α binding motif and the genes harboring the potential HIF-1α binding sites from ChIP-seq database (Rouillard et al., 2016;Zhang et al., 2020a), we identified 27 genes as potential HHRGs in hMSCs (Fig. 5A and 5B).Among these, 24 genes have been identified as HIF-1α target genes by other studies (Lee et al., 2004;Masoud and Li, 2015), while the other three genes were unreported and therefore referred to as novel HHRGs in hMSCs (Fig. 5B).Consistently, we found that hypoxia-induced upregulation of ANKZF1 was abolished upon silencing HIF-1α both by RT-qPCR and Western blot analyses (Fig. 5C and 5D).ANKZF1, encoding ankyrin repeat and zinc finger domain-containing protein 1, was reported to play a role in the cellular response to hydrogen peroxide and in the maintenance of mitochondrial integrity under cellular stress conditions (van Haaften-Visser et al., 2017).To evaluate whether HIF-1α is capable of binding to the predicted four sites of the ANKZF1 promoter, we performed chromatin immunoprecipitation (ChIP)-qPCR with an anti-HIF-1α antibody.Interestingly, we observed specific binding between HIF-1α and the ANKZF1 promoter in HIF-1α +/+ hMSCs relative to HIF-1α −/− cells (Fig. 5E).Subsequently, to query whether ANKZF1

Protein & Cell
is directly activated by HIF-1α in hMSCs, we cloned the ANKZF1 promoter region containing the four putative HIF-1α binding motifs upstream of the luciferase reporter, and found that the promoter of ANKZF1 was indeed transcriptionally activated by hypoxia-induced HIF-1α (Fig. 5F).By contrast, we observed diminished ANKZF1 promoter activity upon mutations of two core base pairs within each predicted binding sites of the ANKZF1, in particular within the site 4, as reflected by a massive reduction of luciferase activity (Fig. 5F).Overall, those data support a role for HIF-1α in positively regulating ANKZF1 transcription in hMSCs.
Finally, we investigated whether ANKZF1 mediates the angiogenic functions of HIF-1α in hMSCs by silencing ANKZF1 via CRISPR/Cas9-mediated knockout system (Fig. 5G).A decrease in protein level of ANKZF1 was confirmed by Western bloting of hMSCs transduced with ANKZF1-targeting sgRNA (Fig. 5H).Indeed, we observed a diminished capacity in cellular migration and in tube formation in ANKZF1-knockout hMSCs (Fig. 5I and 5J), which resembled the phenotypes we had observed in HIF-1α-depleted hMSCs (Fig. 2L and 2M).Collectively, our data suggested that ANKZF1 is a novel target gene of HIF-1α that at least partially contributes to angiogenic modulation in hMSCs.

Discussion
Ischemic conditions that reduce the supply of oxygen and nutrients can lead to severe injury, requiring vascular repair and blood flow recovery processes.However, the molecular programs intrinsic to human vascular cells that partake in ischemic vascular remodeling remain largely unknown.Herein, by using CRISPR/Cas9-mediated genome editing in human ESCs and directed differentiation, we generated HIF-1α-deficient human vascular cells to explore the effects of HIF-1α deficiency on neovascularization.We found that elevated expression of HIF-1α under hypoxic condition augments the angiogenic capability of human vascular cells in vitro and boosts the blood flow recovery at ischemic sites in vivo.We also unveiled that ANKZF1, by acting as a HIF-1α target gene in hMSCs, mediates the pro-angiogenic effect of HIF-1α.In sum, this study adds a layer to our understanding of the role of HIF-1α in human vascular cell homeostasis and angiogenesis, and identifies a new and potentially targetable mechanism for development of therapeutic interventions against ischemic diseases (Fig. 5K).
Ischemic diseases are often caused by blocked blood flow and associated with excessively high morbidity and mortality (Lei et al., 2021;Golledge, 2022).In the past, accumulating studies that used drugs or angiogenic factors to induce neovascularization generated disparate outcomes (Amsden, 2011).However, the majority were found to have limited effectiveness and undesirable side effects (Annex and Cooke, 2021).Therefore, efforts towards molecular profiling in human vascular cell models and in-depth mechanistic analysis aimed at decoding angiogenesis in human tissues are of both scientific and clinical importance.In the present study, we combined CRISPR/Cas9-mediated gene editing technology in hESCs cells with directed differentiation to establish human vascular cell models with genetic manipulation of HIF-1α.By generating this valuable experimental platform, we were able to functionally investigate causal mechanism underlying human vascular cell homeostasis and vascular regeneration, laying the groundwork for development of therapeutic treatments against ischemic diseases.
HIF-1α is generally considered to respond to oxygen level alterations and facilitate adaptation to hypoxia, oxidative stress, and metabolic changes by activating downstream genes (Zheng et al., 2022).However, the involvement of HIF-1α and its downstream molecular mechanism in different types of human vascular cells have not been fully explored.Here, by combining human pluripotent stem cell-derived human vascular cell and hindlimb ischemia mouse models, we uncovered how vascular regeneration and repair, normally supported by various human vascular cells, were abolished by HIF-1α deficiency in vitro and in vivo.Notably, with the exception of delayed restoration of blood flow, the persistent inflammatory responses reflected by enhanced numbers of CD45 + immune cells and elevated cytokine expression (e.g., TNF-α) in the ischemic zone were also observed in the HIF-1α −/− implanted groups.In support of our findings, previous studies demonstrated that HIF-1α overexpression enhances immunomodulation ability by impairing dendritic cell differentiation, inducing suppressor macrophages, and enhancing resistance to NK cell-mediated lysis (Martinez et al., 2017;Cowman and Koh, 2022).Here, we revealed a crucial role of HIF-1α in directing angiogenic capacity of transplanted human vascular cells, thereby modulating the immune microenvironment in vivo, further demonstrating a potential causality between ischemia and inflammation, and supporting a potential therapeutic countermeasure against human ischemic diseases.
Numerous studies of blood vessel have mainly focused on endothelial cells and smooth muscle cells; however, the functions and mechanisms of vascular adventitia have remained understudied.In more recent work, MSCs were reported to reside within the tunica adventitial niche and to instruct vascular morphogenesis, repair, and self-renewal of vascular wall cells, processes that contribute to the local neovascularization in disease processes (Worsdorfer et al., 2017;Klein, 2020;Wang et al., 2022c).Here, based on RNA-seq data, we identified hMSC as the most sensitive cell type to hypoxia and HIF-1α deficiency compared to hVEC and hVSMC.Importantly, our ChIP-qPCR and luciferase reporter analysis support that

Protein & Cell
ANKZF1 is a novel HIF-1α target gene.ANKZF1, a cofactor binding to p97 (Stapf et al., 2011), was found to play a pivotal role in cellular response to hydrogen peroxide and in the maintenance of mitochondrial integrity under conditions of cellular stress (van Haaften-Visser et al., 2017).Here, we discovered that knockdown of ANKZF1 in hMSCs mimicked the impaired angiogenetic phenotypes of HIF-1α −/− hMSC under hypoxia.In support of our observation, a previous study showed that ANKZF1 plays an important role in angiogenesis in colon cancer (Zhou et al., 2019a).Collectively, our findings suggest that ANKZF1 serves as a downstream effector of HIF-1α and contributes to neovascularization in hMSCs.
In summary, we here, for the first time, generated HIF-1α-deficient models of the three major human vascular cells.Through the application of this valuable platform, we unraveled how HIF-1α-associated transcriptional programs boost angiogenesis, and identified ANKZF1 as a novel HIF-1α target gene in human vascular cells.The new pathways and potential targets discovered in this study may facilitate development of new therapeutic approaches for ischemic diseases.

Teratoma assay
Teratoma assays were performed as previously described (Hu et al., 2020).In brief, ~5 × 10 6 hESCs were injected into the groin cavities of NOD/SCID mice (male, 8 weeks old).After ~2 months, the teratomas were collected and analyzed by immunofluorescence staining with indicated markers.

Mouse hindlimb ischemia model induction and cell transplantation
BALB/c nude mice (8-10 weeks old) were used for hindlimb ischemia model construction as previous described (Yan et al., 2019).Briefly, mouse was anesthetized with isoflurane delivered at 2%.The proximal and distal femoral artery of the right hindlimb was ligated using 7-0 nonabsorbable suture.After surgery, 3 × 10 6 hVECs + hVSMCs (3:1) or hMSCs were injected into the ischemic hindlimb in a 100 μL PBS and injected at six different locations immediately.For the control group, 100 μL of PBS without cells was injected.Blood perfusion was monitored every four days by the laser doppler blood perfusion (Moor instruments).Sixteen days after the ligation, hindlimb muscles were harvested for section staining.

Generation of hVECs via directed differentiation from hESCs
hESCs were picked on Matrigel-coated plates and cultured in mTeSR medium.For directed differentiation to hVECs, hESCs were cultured in M1 medium containing IWP2 (3 mmol/L), BMP4 (25 ng/mL), CHIR99021 (3 mmol/L), and FGF2 (4 ng/mL), for 3 days.On the fourth day, M2 medium containing VEGF (50 ng/mL), FGF2 (20 ng/ mL) and IL-6 (10 ng/mL) was used for another 3 days.The differentiated cells were harvested using Accumax and purified with hVEC specific markers (CD201 and CD144) by FACS.Dual-positive cells were collected as hVECs for future experiments.The antibody information was listed in Table S2.

Generation of hVSMCs via directed differentiation from hESCs
hESCs were picked on Matrigel-coated plates and cultured in mTeSR medium for 4-5 days.The hESC clone with high quality was dissociated into single cells using TrypLE and seeded on Matrigel-coated plates with a concentration of 3 × 10 4 cells/cm 2 .On the next day, culture medium was switched to M1 (VSMC basal medium with

Protein & Cell
25 ng/mL BMP4 and 8 μmol/L CHIR99021).On day 3, medium was switched to M2 (VSMC basal medium with 2 ng/mL Activin A and 10 ng/mL PDGF).On day 5, the cells were purified with CD140b antibody by FACS and cultured in VSMC basal medium with 10 ng/mL PDGF for future experiments.The antibody information was listed in Table S2.

RNA extraction and analyses
Total RNA was extracted using TRIzol Reagent.One microgram of total RNA was reverse-transcribed to cDNA by using the GoScript Reverse Transcription System and oligo (dT) primer.PCR was carried out using Taq DNA Polymerase to detect the expression of pluripotency markers OCT4, SOX2, and NANOG in HIF-1α +/+ and HIF-1α −/− hESCs.Human GAPDH was used as an internal control.qPCR was performed using a CFX384 Real-Time PCR system with iTaq Universal SYBR Green Super mix to verify the transcript changes of predict HIF-1α target genes.Human β-actin was used as an internal control.Primers used in this study are listed in Table S3.

Immunofluorescence staining
Samples of cells seeded on coverslip or OCT embedding tissue sections were fixed in 4% paraformaldehyde, permeabilized in 0.4% Triton X-100 and blocked in 5% BSA-PBS.Primary antibodies were diluted in blocking buffer (5% BSA-PBS) and an incubation was conducted overnight at 4°C.After removal of the extra primary antibodies by PBS washing, samples were incubated with the corresponding fluorescence-labeled secondary antibodies at room temperature for 1 h.Nuclear DNA was labeled by Hoechst 33342.The fluorescent-positive cells or tissues were captured by laser scanning confocal microscopy and quantified using Image J software.The antibody information was listed in Table S2.

Western blot
To detect the protein levels of HIF-1α and ANKZF1, cells were harvested in 2% SDS (w/v) solution supplemented with protease inhibitor cocktail (Roche) and boiled for 10 min.Protein concentration was measured by a BCA protein assay kit (Bicinchoninic acid).Twenty microgram total protein was loaded into SDS-PAGE gels for protein separation and then electro-transferred to PVDF membranes (Millipore).Following blocking with 5% (w/v) nonfat powdered milk (BBI Life Sciences) for 1 h at room temperature, the membrane was incubated with the corresponding primary antibodies overnight at 4°C.Then, the membrane was washed by TBST and incubated by HRP-conjugated respective secondary antibodies at room temperature for 1 h.Finally, image was generated by Image Lab 3.0 software (Bio-Rad) and analyzed with relative gray value by image J.The antibody information was listed in Table S2.

ChIP-qPCR
ChIP-qPCR was performed according to previous protocols with slight modifications (Hu et al., 2020).Briefly, 1 × 10 6 hMSCs pretreated with 3% O 2 for 48 h were crosslinked by 1% (v/v) formaldehyde diluted in PBS for 13 min.The reaction was stopped by an incubation in 0.125 mol/L Glycine for 5 min at room temperature.After washes with PBS, cells were resuspended in ice-cold lysis buffer (50 mmol/L Tris-HCl, 10 mmol/L EDTA, 1% SDS, pH 8.0) for 5 min.After sonication by a Bioruptor® Plus device (Diagenode), supernatants were incubated overnight at 4°C with Protein A/G dynabeads (Thermo Fisher Scientific, 10004D) conjugated with anti-HIF-1α, or normal rabbit IgG.Subsequently, elution and reverse cross-linking were performed at 68°C for 3 h on Protein & a thermomixer.DNA was then isolated by the phenolchloroform-isoamylalcohol extraction and ethanol precipitation method, and the purified DNA was used for qPCR detection.Primers used in this study are listed in Table S3.

Plasmid construction and luciferase reporter assay
The promoter region (2,000 bp upstream of the transcription start site of ANKZF1) was obtained via PCR amplification and then cloned into PGL3-basic vector.The plasmids carrying the mutations of the binding sites within the promoter of ANKZF1 were constructed using a Fast MultiSite Mutagenesis System (Transgen, Cat.No# FM201) and the mutagenic primers according to the manufacturer's instructions.For single binding site mutation (Mut1, Mut2, Mut3, and Mut4), the corresponding primer pair was used; for multiple binding site mutation (Mut1-4), four pairs of primer were used together for amplification.The mutations were confirmed by DNA sequencing.Primers used in this study are listed in Table S3.
For luciferase reporter assay, hMSCs were cultured in 24-well plates and co-transfected with 1.0 μg plasmid of luciferase driven by ANKZF1 promoter and 0.2 μg plasmid carrying Renilla using Lipofectamine® 3000 (Invitrogen).Forty-eight hours after transfection, cells were collected and relative luciferase activity was measured using Dual-Luciferase Reporter Assay System (T002, Vigorous Biotechnology Beijing Co., Ltd.).

Transwell migration assay
For the transwell migration assay, 2 × 10 4 cells were seeded on the top of 0.8 μm filters (Costar) in basal medium.Then, filters were placed into 24 culture plate wells containing complete medium.After 24 h of culture for hMSCs and hVECs or 48 h for hVSMCs, the filter inserts were fixed with 4% paraformaldehyde and then were stained by crystal violet for 30 min at room temperature.After washing, the migrated cells were photographed by light microscope and counted with Image J.

In vitro tube formation assay
For the tube formation assay, 6 × 10 4 cells were suspended in 600 μL complete medium and then seeded on Matrigel-coated 24-well plate.After 8-12 h, lattice-like vessel structures formed and the cells were then incubated with Calcein-AM (HY-D0041, Med Chem Express LLC) and examined by using fluorescence microscope.

Masson's trichrome staining
Ischemic hindlimb sections were washed three times with PBS, and then stained according to the protocol of Masson's Trichrome stain kit (G1340, Solarbio).All images were captured using a digital pathology slide scanner (Aperio CS2, Leica).Infarcted scar size was calculated by using image J.

CNV analysis
The genomic DNA was isolated from 1 × 10 6 HIF-1α +/+ or HIF-1α −/− hESCs by using a DNeasy Blood & Tissue Kit (Qiagen).Quality control and sequencing were performed following standard protocols from Novogene Bioinformatics Technology Co. Ltd.Genome-wide CNV analysis was conducted as previously described (Yan et al., 2019).Raw reads were trimmed by the Trim Galore software (version 0.5.0) and clean reads were aligned to the UCSC hg19 human genome using bowtie2 software (version 2.2.9) (Langmead and Salzberg, 2012).R package HMMcopy (version 1.28.1) was implemented to calculate CNVs in each 0.5 Mb bin size (Ha et al., 2012).

RNA-seq library construction and sequencing
Using the NEBNext® Poly (A) mRNA Magnetic Isolation Module, mRNA was isolated for RNA-seq.We constructed sequencing libraries using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina following the manufacturer's protocol.The libraries were sequenced on Illumina HiSeq X-Ten platforms with paired-end 150-bp sequencing.Quality control and RNA sequencing were done by Novogene Bioinformatics Technology.

RNA-seq data processing
Raw data were trimmed by Trim Galore software (version 0.5.0).Clean data were mapped to the human reference genome (hg19) by HISAT2 software (version 2.0.4) (Kim et al., 2015).The reads mapped to gene were calculated using HTSeq software (version 0.11.0)(Anders et al., 2015).DEGs were calculated using the DEseq2 (version 1.30.1)(Love et al., 2014) with the cutoff of adjust P value less than 0.05 and |log 2 (fold change)| more than 0.5.The FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) of the gene was calculated using StringTie software (Pertea et al., 2015).GO terms and pathways enrichment analysis were performed by Metascape (Zhou et al., 2019b).The motif of HIF-1α was drawn using data from the JASPAR database (Castro-Mondragon et al., 2022).The predicted binding sites of HIF-1α on promoter of target genes were screened using MEME's "motif scanning" function (Bailey and Elkan, 1994).3 kb upstream of transcription start site was selected as promoter region.The DEGs are listed in Table S1.

Statistical analysis
Data are shown as the mean ± SEM.Two-tailed Student's t test was used for comparing the difference between groups.Multiple group comparisons were performed by one-way ANOVA followed by Tukey's test or two-way ANOVA followed by Sidak's test.GraphPad Prism 8.0 was used for statistical analysis.P < 0.05 is considered statistically significant.

Figure 4 .
Figure 4. Transcriptomic analysis reveals the role of HIF-1α in human vascular cells.(A) Wind rose plots showing DEGs numbers between HIF-1α +/+ cells (hVECs, hVSMCs, and hMSCs) under normoxic and hypoxic condition (left), or between HIF-1α +/+ and HIF-1α −/− cells (hVECs, hVSMCs, and hMSCs) under hypoxic condition (right).(B) Venn diagrams showing the number of upregulated hypoxiaassociated DEGs and the number of downregulated DEGs in human vascular cells with HIF-1α deficiency.The number of indicated overlapping genes was also shown and defined as "hypoxia-induced HIF-1α responsive genes (HHRGs)".(C) GO term and pathway enrichment analysis of HHRGs across different human vascular cells.(D) Bubble plot showing the relative expression levels of DEGs associated with indicated terms and pathways enriched in panel (C).(E) Venn diagram showing the number of overlapping genes or cell type-specific genes of HHRGs in hVECs, hVSMCs, and hMSCs.(F) Heatmaps showing the relative expression levels of DEGs in two or three cell types.(G) Dot plots showing the relative expression levels of HHRGs in hVECs, hVSMCs, and hMSCs.

Figure 5 .
Figure 5. ANKZF1 acts as a major effector gene downstream of HIF-1α in hMSCs.(A) Conjoint analysis of HHRGs containing the canonical HIF-1α binding motif and the genes harboring the potential HIF-1α binding sites from ChIP-seq database.(B) 27 overlapped genes in panel (A) were exhibited as potential HHRGs in hMSCs by network plot diagram.Three genes with orange background were referred to as novel HHRGs in hMSCs (C) RT-qPCR verified the changes of ANKZF1 mRNA level upon HIF-1α depletion in hMSCs.Data are shown as the mean ± SEM. n = 3 independent experiments.One-way ANOVA followed by Tukey's test.(D) Western blot analysis of ANKZF1 upon HIF-1α depletion in hMSCs.Data are shown as the mean ± SEM. n = 3 independent experiments.One-way ANOVA followed by Tukey's test.(E) ChIP-qPCR analysis showing the binding of HIF-1α to ANKZF1 promoter.Data were presented as mean ± SEM.Two-tailed Student's t test.(F) Diagram (left) and quantitative data (right) of luciferase reporter assay of HIF-1α on the luciferase activity expressed from ANKZF1 promoter.Data are shown as the mean ± SEM. n = 3 independent cell culture wells.One-way ANOVA followed by Tukey's test.(G) Schematic diagram showing the CRSIPR/Cas9-mediated knockout of ANKZF1 in hMSCs and subsequent related experiments.(H) Western blot showing the protein level of ANKZF1 in hMSCs transduced with nontargeting (sg-NTC) or ANKZF1 targeting sgRNA.Left, representative images of Western Right, statistical analysis of relative protein levels of ANKZF1.β-actin was used as the loading control.Data are presented as the mean ± SEM, n = 3 independent experiments.Twotailed Student's t test.(I) Representative images and quantitative data of cell migration assay in hMSCs transduced with sg-NTC or sg-ANKZF1 sgRNA.Scale bars, 200 μm.n = 3 independent cell culture wells.Data are shown as the mean ± SEM.Student's t test.Representative images and quantitative data of tube formation assay in hMSCs transduced with sg-NTC or sg-ANKZF1 sgRNA.Scale bars, 500 μm.n = 3 independent cell culture wells.Data are shown as the mean ± SEM.Two-tailed Student's t test.(K) A schematic illustration showing the effects of HIF-1α on human vascular in vitro and in vivo, and identified ANKZF1 as a novel HIF-1α target gene in human cells.