Vasoinhibin’s Apoptotic, Inflammatory, and Fibrinolytic Actions Are in a Motif Different From Its Antiangiogenic HGR Motif

Vasoinhibin, a proteolytic fragment of the hormone prolactin, inhibits blood vessel growth (angiogenesis) and permeability, stimulates the apoptosis and inflammation of endothelial cells, and promotes fibrinolysis. The antiangiogenic and antivasopermeability properties of vasoinhibin were recently traced to the HGR motif located in residues 46 to 48 (H46-G47-R48), allowing the development of potent, orally active, HGR-containing vasoinhibin analogues for therapeutic use against angiogenesis-dependent diseases. However, whether the HGR motif is also responsible for the apoptotic, inflammatory, and fibrinolytic properties of vasoinhibin has not been addressed. Here, we report that HGR-containing analogues are devoid of these properties. Instead, the incubation of human umbilical vein endothelial cells with oligopeptides containing the sequence HNLSSEM, corresponding to residues 30 to 36 of vasoinhibin, induced apoptosis, nuclear translocation of NF-κ B, expression of genes encoding leukocyte adhesion molecules ( VCAM1 and ICAM1 ) and proinflammatory cytokines ( IL1B, IL6, and TNF ), and adhesion of peripheral blood leukocytes. Also, intravenous or intra-articular injection of HNLSSEM-containing oligopeptides induced the expression of Vcam1, Icam1, Il1b, Il6, and Tnf in the lung, liver, kidney, eye, and joints of mice and, like vasoinhibin, these oligopeptides promoted the lysis of plasma fibrin clots by binding to plasminogen activator inhibitor-1 (PAI-1). Moreover, the inhibition of PAI-1, urokinase plasminogen activator receptor, or NF-κ B prevented the apoptotic and inflammatory actions. In conclusion, the functional properties of vasoinhibin are segregated into 2 different structural determinants. Because apoptotic, inflammatory, and fibrinolytic actions may be undesirable for antiangiogenic therapy, HGR-containing vasoinhibin analogues stand as selective and safe agents for targeting pathological angiogenesis.

The formation of new blood vessels (angiogenesis) underlies the growth and repair of tissues and, when exacerbated, contributes to multiple diseases, including cancer, vasoproliferative retinopathies, and rheumatoid arthritis (1).Antiangiogenic therapies based on tyrosine kinase inhibitors (2,3) and monoclonal antibodies against vascular endothelial growth factor (VEGF) or its receptor (4) have proven beneficial for the treatment of cancer and retinal vasoproliferative diseases (5).However, disadvantages such as toxicity (6)(7)(8) and resistance (9) have incentivized the development of new treatments.
Moreover, vasoinhibin, by itself, activates the NF-κB pathway in endothelial cells to stimulate apoptosis (11) and trigger the expression of inflammatory factors and adhesion molecules, resulting in leukocyte infiltration (12).Finally, vasoinhibin promotes the lysis of a fibrin clot by binding to PAI-1 and inhibiting its antifibrinolytic activity (13).
The antiangiogenic determinant of vasoinhibin was recently traced to a short linear motif of just 3 amino acids (His46-Gly47-Arg48) (the HGR motif), which led to the development of heptapeptides comprising residues 45 to 51 of vasoinhibin that inhibited angiogenesis and vasopermeability with the same potency as whole vasoinhibin (14) (Fig. 1A).The linear vasoinhibin analogue (Vi45-51) was then optimized into a fully potent, proteolysisresistant, orally active cyclic retro-inverse heptapeptide (CRIVi45-51) (Fig. 1A) for the treatment of angiogenesisdependent diseases (14).Notably, thrombin generates a vasoinhibin of 48 amino acids (Vi1-48) that contains the HGR motif (Fig. 1A).Vi1-48 is antiangiogenic and fibrinolytic (15), suggesting that the HGR motif could also be responsible for the apoptotic, inflammatory, and fibrinolytic properties of vasoinhibin.This possibility needed to be analyzed to support the therapeutic future of the HGR-containing vasoinhibin analogues as selective and safe inhibitors of blood vessel growth and permeability.Moreover, the identification of specific functional domains within the vasoinhibin molecule provides insights and tools for understanding its overlapping roles in angiogenesis, inflammation, and coagulation under health and disease.

Cell Invasion
HUVEC invasion was evaluated using the Transwell Matrigel barrier assay (21).HUVEC were seeded at 28 000 cells cm −2 on the luminal side of an 8-µm-pore insert of a 6.The medium of HUVEC was replaced with 100 μL of Hoechst-stained leukocytes (10 5 leukocytes per well) and incubated for 1 hour at 37 °C.Finally, HUVEC were washed 3 times with warm PBS, and images were obtained in an inverted fluorescent microscope (Olympus IX51) and quantified using the CellProfiler software (20).
Cells were incubated at room temperature for 30 minutes and centrifugated at 20 000g for 10 minutes (Avanti J-30I Centrifuge, Beckman Coulter, Brea, CA).The supernatant was collected and diluted 1:5 with incubation buffer (final concentration ∼20 000 cells mL −1 ).HUVEC concentration was standardized, and the assay was carried out according to the manufacturer's instructions, measuring absorbance at 415 nm.
These findings show that the HGR-containing vasoinhibin analogues lack the apoptotic, inflammatory, and fibrinolytic properties of vasoinhibin.The fact that PRL is not inflammatory, apoptotic, or fibrinolytic indicates that, like the antiangiogenic effect (14), these vasoinhibin properties emerge upon PRL cleavage.

HGR-Containing Vasoinhibin Analogues Do Not Stimulate the Nuclear Translocation of NF-κB and the Expression of Inflammatory Molecules in HUVEC
Because vasoinhibin signals through NF-κB to induce the apoptosis and inflammation of endothelial cells (11,12), we asked whether HGR-containing vasoinhibin analogues were able to promote the nuclear translocation of NF-κB and the expression of proinflammatory mediators in HUVEC (Fig. 2).The distribution of NF-κB in HUVEC was studied using fluorescence immunocytochemistry and monoclonal antibodies against the p65 subunit of NF-κB (Fig. 2A).Without treatment, p65 was homogeneously distributed throughout the cytoplasm of cells.Treatment with Vi1-123 or Vi1-48, but not with Vi45-51, CRIVi45-51, or PRL, resulted in the accumulation of p65 positive stain in the cell nucleus (Fig. 2A) indicative of the NF-κB nuclear translocation/activation needed for transcription.Consistently, only vasoinhibin isoforms (Vi1-123 or Vi1-48) and not the HGR-containing vasoinhibin analogues nor PRL induced the mRNA expression of genes encoding leukocyte adhesion molecules (intercellular adhesion molecule 1 [ICAM1] and vascular cell adhesion molecule 1 [VCAM1]) and proinflammatory cytokines (IL-1α (IL1A), IL-1β (IL1B), IL-6 (IL6), and tumor necrosis factor α [TNF]) in HUVEC (Fig. 2B).These findings show that HGR-containing vasoinhibin analogues are unable to activate NF-κB to promote gene transcription, resulting in the apoptosis and inflammation of HUVEC.Furthermore, these results suggest that a structural determinant-different from the HGR motif-is responsible for these properties.

In Vivo Inflammation Is Stimulated by the HNLSSEM Sequence and Not by the HGR Motif
To evaluate whether the HGR or the HNLSSEM motifs promotes the inflammatory phenotype of endothelial cells in vivo, the HGR-containing vasoinhibin analogue Vi45-51 or the HNLSSEM-containing 30-45 oligopeptide was injected intravenously to reach an estimated ≃10 μM concentration in serum, and after 2 hours, mice were perfused, and lung, liver, kidney, and eyes were collected to evaluate mRNA expression of leukocyte adhesion molecules (Icam1 and Vcam1) and cytokines (Il1b, Il6, and Tnf), and the level of leukocyte marker (Cd45).The underlying rationale is that intravenous delivery and short-term (2-hour) analysis in thoroughly perfused animals would reflect a direct effect of the treatments on endothelial cell mRNA expression of inflammatory factors in the various tissues.The 30-45 peptide, but not the Vi45-51, increased the expression levels of these inflammatory markers in the evaluated tissues (Fig. 5A-5D).Furthermore, because vasoinhibin is inflammatory in joint tissues (24), we injected into the knee cavity of mice 87 pmol of the Vi45-51 or the 30-45 peptide, and after 24 hours, only the 30-45 oligopeptide induced the mRNA expression of Il1b, Il6, and inducible nitric oxide synthetase (Inos) (Fig. 5E).The finding in joints implied that, like vasoinhibin, the inflammatory effect of the 30-45 peptide extends to other vasoinhibin target cells, that is, synovial fibroblasts (24).

Discussion
Vasoinhibin represents a family of proteins comprising the first 48 to 159 amino acids of PRL, depending on the cleavage site of several proteases, including matrix metalloproteases (26), cathepsin D (27), bone morphogenetic protein 1 (28), thrombin (15), and plasmin (29).The cleavage of PRL occurs at the hypothalamus, the pituitary gland, and the target tissue levels, defining the PRL/vasoinhibin axis (30).This axis contributes to the physiological restriction of blood vessels in ocular (31,32) and joint (26) tissues and is disrupted in angiogenesis-related diseases, including diabetic retinopathy (33), retinopathy of prematurity (34), peripartum cardiomyopathy (35), preeclampsia (36), and inflammatory arthritis (37).Furthermore, 2 clinical trials have addressed vasoinhibin levels as targets of therapeutic interventions (38).However, the clinical translation of vasoinhibin is limited by difficulties in its production (39).These difficulties were recently overcome by the development of HGR-containing vasoinhibin analogues that are easy to produce, as well as potent, stable, and even orally active to inhibit the growth and permeability of blood vessels in experimental vasoproliferative retinopathies and cancer (14).Nonetheless, the therapeutic value of HGR analogues is challenged by evidence showing that vasoinhibin is also apoptotic, inflammatory, and fibrinolytic, properties that may worsen microvascular diseases (40,41).Here, we show that the various functions of vasoinhibin are segregated into 2 distinct, nonadjacent, and independent small linear motifs: the HGR motif responsible for the vasoinhibin inhibition of angiogenesis and vasopermeability (14) and the HNLSSEM motif responsible for the apoptotic, inflammatory, and fibrinolytic properties of vasoinhibin (Fig. 7A).The HGR and HNLSSEM motifs are inactive in PRL, the vasoinhibin precursor.We confirmed that PRL has no antiangiogenic properties (10) and showed that PRL lacks apoptotic and inflammatory actions on endothelial cells as well as no fibrinolytic activity.PRL has 199 amino acids structured into a 4-⍺-helix bundle topology connected by 3 loops (43).The HGR motif is in the first part of loop 1 (L1) connecting ⍺-helixes 1 and 2, whereas the HNLSSEM motif is in ⍺-helix 1 (H1) (Fig. 7A).Upon proteolytic cleavage, PRL loses its fourth ⍺-helix (H4), which drives a conformational change and the exposure of the HGR motif, obscured by H4 (14,42).Since H1 and H4 are in close contact in PRL (43), it is likely that some elements of H4 also mask the HNLSSEM motif.Alternatively, it is also possible that residues of the HNLSSEM motif buried in the hydrophobic core of PRL become solvent exposed by the conformational change into vasoinhibin.However, this is unlikely since the hydrophobic core appears conserved during vasoinhibin generation (42).
A previous report indicated that binding to PAI-1 mediates the antiangiogenic actions of vasoinhibin (23).Contrary to this claim, antiangiogenic HGR-containing vasoinhibin analogues did not bind PAI-1, whereas the HNLSSEM-oligopeptides bound PAI-1 but did not inhibit HUVEC proliferation and invasion.While these findings unveil the structural determinants in vasoinhibin responsible for PAI-1 binding, they question the role of PAI-1 as a necessary element for the antiangiogenic effects of vasoinhibin.Little is known of the molecular mechanism by which vasoinhibin binding to the PAI-1-uPA-uPAR complex inhibits endothelial cells (23).Although the binding could help localize vasoinhibin on the surface of endothelial cells, the contribution of other vasoinhibin-binding proteins and/or interacting molecules cannot be excluded.For example, integrin ⍺5β1 interacts with the uPA-uPAR complex (44), and vasoinhibin binds to ⍺5β1 to promote endothelial cell apoptosis (45).Nevertheless, none of the HGR-containing analogues induced apoptosis.Therefore, the binding molecule/receptor that transduces the antiangiogenic properties of vasoinhibin remains unclear.
Vasoinhibin is commonly described as antiangiogenic due to its ability to inhibit endothelial cell proliferation, migration, and survival.However, the proapoptotic effect of vasoinhibin can occur independent of its antiangiogenic action.For example, vasoinhibin contributes to the physiological regression by apoptosis of the stable hyaloid vasculature, a transient network of intraocular vessels that nourishes the immature lens, retina, and vitreous (46).Moreover, despite lacking proapoptotic properties, Vi45-51 inhibits the growth of melanoma tumors ( 14) like whole vasoinhibin (23,(47)(48)(49), to suggest that the apoptotic effect of vasoinhibin may be irrelevant to its overall antiangiogenic efficacy.
Consistent with previous reports (11,12,23), vasoinhibin binding to PAI-1 and activation of uPAR and NF-κB did associate with the apoptotic, inflammatory, and fibrinolytic properties of the HNLSSEM-containing oligopeptides.These oligopeptides, but not HGR-containing oligopeptides, induced endothelial apoptosis, nuclear translocation of NFκB, expression of leukocyte adhesion molecules and proinflammatory cytokines, and adhesion of leukocytes, as well as the lysis of plasma fibrin clot.The inflammatory action, but not the apoptotic effect, was prevented by PAI-1 immunoneutralization, whereas both inflammatory and apoptotic actions were blocked by anti-uPAR antibodies or by an inhibitor of NF-κB.Locating the apoptotic, inflammatory, and fibrinolytic activity in the same short linear motif of vasoinhibin is not unexpected since the 3 events can be functionally linked.The degradation of a blood clot is an important aspect of inflammatory responses, and major components of the fibrinolytic system are regulated by inflammatory mediators (50).Examples of such interactions are the thrombin-induced generation of vasoinhibin during plasma coagulation to promote fibrinolysis (15), the endotoxin-induced IL-1 production inhibited by PAI-1 (51), and the TNFα-induced suppression of fibrinolytic activity due to the activation of NFκB-mediated PAI-1 expression (52).Furthermore, uPA is upregulated by thrombin and inflammatory mediators in endothelial cells (53), and uPAR is elevated under inflammatory conditions (54).On the other hand, the identification of HNLSSEM as the motif responsible for the binding of vasoinhibin to PAI-1 raises the possibility that such motif contributes to the interaction of other proteins with PAI-1, like vitronectin, uPA, and tPA (55).Our preliminary analyses found no identical HNLSSEM sequences in these proteins but detected similar motifs in regions shown to be irrelevant for binding to PAI-1 that merit further research.The HNLSSEM-oligopeptides' inflammatory action is further supported by their in vivo administration.The intravenous injection of HNLSSEM-oligopeptides upregulated the short-term (2 hours postinjection) expression of Icam1 and Vcam1, IlIb, Il6, and Tnf, and the infiltration of leukocytes (evaluated by the expression levels of the leukocyte marker Cd45) in different tissues indicative of an inflammatory action on different vascular beds.Also, the HNLSSEM-oligopeptides injected into the intra-articular space of joints launched a longer-term inflammation (24 hours postinjection) indicative of an inflammatory response in joint tissues.This action is consistent with the vasoinhibin-induced stimulation of the inflammatory response of synovial fibroblasts, primary effectors of inflammation in arthritis (37).
The challenge is to understand when and how vasoinhibin impacts angiogenesis, apoptosis, inflammation, and fibrinolysis pathways under health and disease.One likely example is during the physiological repair of tissues after wounding and inflammation.By inhibiting angiogenesis, vasoinhibin could help counteract the proangiogenic action of growth factors and cytokines, whereas by stimulating apoptosis, inflammation, and fibrinolysis, vasoinhibin could promote the pruning of blood vessels, protective inflammatory reactions, and clot dissolution needed for tissue remodeling.Indeed, the antiangiogenic effect of vasoinhibin can be accompanied by its inflammatory actions.Although, the HNLSSEM motif has 5.3 times less potency than the HGR motif (Fig. 7B).However, in the absence of successful containment, overproduction of blood vessels, persistent inflammation, and dysfunctional coagulation determines the progression and therapeutic outcomes in cancer (41,56), diabetic retinopathy (57), and rheumatoid arthritis (58).The complexity of vasoinhibin actions under disease is exemplified in murine antigen-induced arthritis, where vasoinhibin ameliorates pannus formation and growth via an antiangiogenic mechanism but promotes joint inflammation by stimulating the inflammatory response of synovial fibroblasts (37,59).
Antiangiogenic drugs, in particular VEGF inhibitors, have reached broad usage in the field of cancer and retinopathy, albeit with partial success and safety concerns (6,60,61).They display modest efficacy and survival times, resistance, and mild to severe side effects that include infections, bleeding, wound healing complications, and thrombotic events.Toxicities illustrate the association between the inhibition of blood vessel growth and multifactorial pathways influencing endothelial cell apoptosis, inflammation, and coagulation (6).The fact that the HGR analogues lack the apoptotic, inflammatory, and fibrinolytic properties of vasoinhibin highlights their future as potent and safe inhibitors of blood vessel growth, avoiding drug resistance through their broad action against different proangiogenic substances.
In summary, this work segregates the activities of vasoinhibin into 2 linear determinants and provides clear evidence that the HNLSSEM motif is responsible for binding to PAI-1 and exerting apoptotic, inflammatory, and fibrinolytic actions via PAI-1, uPAR, and NF-κB pathways, while the HGR motif is responsible for the antiangiogenic effects of vasoinhibin.This knowledge provides tools for dissecting the differential effects and signaling mechanisms of vasoinhibin under health and disease and for improving its development into more specific, potent, and less toxic antiangiogenic, proinflammatory, and fibrinolytic drugs.
CRIVi45-51, or the oligopeptides1-15, 12-25, 20-35,  30-45, or 35-48, diluted in PBS.Microplate was blocked for 1 hour at room temperature with 5% w/v nonfat dry milk in 0.1% Tween-20-PBS (PBST), followed by 3 washes with PBST.Next, 100 nM of PAI-1 diluted in 0.2 mg mL −1 bovine serum albumin (BSA)-PBST was added and incubated for 1 hour at room temperature, followed by a 3-wash step with PBST.Anti-PAI-1 antibodies (1 μg mL −1 diluted in blocking buffer) were added and incubated for 1 hour at room temperature.Microplates were then washed 3 times with PBST, and goat anti-rabbit HRP antibody (Jackson ImmunoResearch Labs, West Grove, PA, Cat# 111-035-144, RRID:AB_ 2307391) at 1:2500 (diluted in 50% blocking buffer and 50% PBS) added and incubated for 1 hour at room temperature.Three last washes were done with PBST and microplates incubated for 30 minutes under darkness with 100 μL per well of an o-phenylenediamine dihydrochloride (OPD) substrate tablet diluted in 0.03% H 2 O 2 citrate buffer (pH 5).Finally, the reaction was stopped with 50 µL of 3 M HCl, and absorbance measured at 490 nm.NF-κB Nuclear Translocation AssayHUVEC were seeded on 1 μg cm −2 fibronectin-coated 18 mm-coverslips placed in 12-well plates and grown in complete media to 80% confluence.Then, cells were treated, HUVEC were washed and incubated with 1:500 goat antimouse secondary antibodies coupled to Alexa fluor 488 (Abcam, Cambridge, UK, Cat# ab150113, RRID: AB_2576208) in 1% BSA, 0.1% Tx-100 PBS (2 hours in darkness).Nuclei were counterstained with 5 μg mL −1 Hoechst 33342 (Sigma-Aldrich).Coverslips were mounted with Vectashield (Vector Laboratories, Burlingame, CA) and digitalized under fluorescence microscopy (Olympus IX51).onds at 95 °C, 30 seconds at the annealing temperature of each primer pair, and 30 seconds at 72 °C.The mRNA expression levels were calculated by the 2 −ΔΔCT method.Animals C57BL6 mice were housed under standard laboratory conditions.Experiments were approved by the Bioethics Research Committee of the Institute of Neurobiology of the National University of Mexico (UNAM) in compliance with the US National Research Council's Guide for the Care and Use of Laboratory Animals (8th ed, National Academy Press, Washington, DC).