S-nitrosoglutathione reductase (GSNOR) enhancesvasculogenesis by mesenchymal stem cellsSamirah A. Gomesa, Erika B. Rangela, Courtney Premera, Raul A. Dulcea, Yenong Caoa, Victoria Floreaa, Wayne Balkana,Claudia O. Rodriguesa,b, Andrew V. Schallyc,d,e,1, and Joshua M. Harea,f,1

aInterdisciplinary Stem Cell Institute, bDepartment of Molecular and Cellular Pharmacology, cDepartment of Pathology, dDivision of Hematology/Oncology,and fDivision of Cardiology, Department of Medicine, Leonard M. Miller School of Medicine, University of Miami, Miami, FL 33136; and eEndocrine,Polypeptide and Cancer Institute, Veterans Affairs Medical Center, Miami, FL 33136

Contributed by Andrew V. Schally, November 27, 2012 (sent for review October 31, 2012)

Although nitric oxide (NO) signaling promotes differentiation andmaturation of endothelial progenitor cells, its role in the differen-tiation of mesenchymal stem cells (MSCs) into endothelial cellsremains controversial. We tested the role of NO signaling in MSCsderived from WT mice and mice homozygous for a deletion ofS-nitrosoglutathione reductase (GSNOR−/−), a denitrosylase thatregulates S-nitrosylation. GSNOR−/− MSCs exhibited markedlydiminished capacity for vasculogenesis in an in vitro Matrigeltube–forming assay and in vivo relative to WT MSCs. This decreasewas associated with down-regulation of the PDGF receptorα(PDGFRα) in GSNOR−/− MSCs, a receptor essential for VEGF-Aaction in MSCs. Pharmacologic inhibition of NO synthase withL-NG-nitroarginine methyl ester (L-NAME) and stimulation of growthhormone–releasing hormone receptor (GHRHR) with GHRH agonistsaugmented VEGF-A production and normalized tube formation inGSNOR−/− MSCs, whereas NO donors or PDGFR antagonist re-duced tube formation ∼50% by murine and human MSCs. Theantagonist also blocked the rescue of tube formation inGSNOR−/− MSCs by L-NAME or the GHRH agonists JI-38, MR-409,and MR-356. Therefore, GSNOR−/− MSCs have a deficient capacityfor endothelial differentiation due to downregulation of PDGFRαrelated to NO/GSNOR imbalance. These findings unravel importantaspects of modulation of MSCs by VEGF-A activation of the PDGFRand illustrate a paradoxical inhibitory role of S-nitrosylation sig-naling in MSC vasculogenesis. Accordingly, disease states charac-terized by NO deficiency may triggerMSC-mediated vasculogenesis.These findings have important implications for therapeutic applica-tion of GHRH agonists to ischemic disorders.

angiogenesis | nitroso–redox imbalance

Nitric oxide (NO) andVEGF signaling promotes vasculogenesisby endothelial progenitor cells (EPCs) (1–5). For example,mice deficient in endothelial NO synthase (NOS3−/−) show re-duced VEGF-induced mobilization of bone marrow progenitorcells to sites of injury (4). In EPC-mediated vasculogenesis, VEGF-Aactivates its major receptor [VEGF receptor 2 (VEGFR2)], trig-gering cell differentiation into mature endothelial cells (ECs) andenhancing angiogenesis (6, 7).Mesenchymal stem cells (MSCs) also participate in postnatal

angiogenesis (8, 9), and vascular pericytes, which are crucial formaintaining vascular integrity, share similar phenotypic featureswith MSCs (10). Exogenously administered, MSCs readily formnew capillaries and medium-sized arteries (11, 12), propertiesimportant for the tissue regenerative capacity of MSCs (13). Weand others have shown that MSCs differentiate into endothelialcells in vitro (14) and in vivo and contribute to neovascularization,particularly during tissue ischemia and tumor vascularization (8,11, 12, 15). As with EPCs, VEGF also plays an important role instimulating MSC differentiation, but does so by activating thePDGF receptor (PDGFR) as opposed to the VEGFR2, which isabsent on MSCs (16). However, the impact of NO signaling in thedifferentiation of MSCs into endothelial cells has not been pre-viously tested. Given the similar signaling involved in endothelial

differentiation of EPCs and MSCs, we reasoned that NO playsan equivalent role in this process.Accordingly, we tested the hypothesis that NO signaling, me-

diated by small molecular weight thiols (molecular weight < 500),promotes MSC differentiation into endothelial cells. To test ourhypothesis, we assessed the functional consequences of deletionof S-nitrosoglutathione reductase (GSNOR), which in turnincreases S-nitrosothiols (17, 18), on MSC-mediated vasculo-genesis. We report that paradoxically, MSCs from GSNOR−/−

mice exhibit diminished endothelial differentiation, therebydemonstrating an inhibitory effect of S-nitrosylation on vascu-logenesis mediated by MSCs.

ResultsMurine BoneMarrowMSCCharacterization.Both WT and GSNOR−/−-derived MSCs were spindle shaped, adherent to plastic tissueculture dishes (Fig. S1A), and negative for Lineage, a mixture ofhematopoietic markers, CD34 and CD45, and positive for stemcell antigens SCA-1, CD73, CD90.2, and CD105 (Fig. S1 B and C).

NO Signaling and MSCs. Denitrosylation is controlled in significantpart by GSNOR, a unique and specific enzyme for which S-nitro-soglutathione (GSNO) is the substrate (18) (Fig. S2A). NeitherMSCs (Fig. S2B andC) nor liver (Fig. S2D) fromGSNOR−/−miceexpresses GSNOR. NOS1 and NOS2 were constitutively expressedin both strains of MSCs (Fig. S2 E and F), whereas NOS3 expres-sion was absent from both strains (Fig. S2G). Interestingly, NOS1mRNA was ∼100-fold higher in GSNOR−/− mice (2.7 × 103 ± 3 ×102 absolute number of transcripts,ΔCt) comparedwithWTMSCs(2× 104± 2 × 103, P< 0.05; Fig. S2E). Despite the up-regulation ofNOS1, actual NO production, measured by 4,5-diamino-fluores-cein diacetate (DAF-2DA), was nearly identical between thestrains (Fig. S2H). Thus, GSNOR deficiency up-regulates NOS1but does not lead to increased NO production.

GSNOR−/− MSCs Exhibit Impaired Formation of Capillary Tube–LikeStructures in Vitro. Next we used a Matrigel assay to analyze theability of MSCs to form tube-like structures in vitro. Cells weregrown in endothelial medium (EGM-2; Lonza) for 1 wk beforeplating on Matrigel. Surprisingly, GSNOR−/−-derived MSCsformed significantly fewer (29.9 ± 11.75 vs. 50.2 ± 14.04, P <0.001) and shorter (62.8 ± 14.0 vs. 124.2 ± 49.0 μm, P < 0.001)

Author contributions: S.A.G., W.B., and J.M.H. designed research; S.A.G., E.B.R., C.P., R.A.D.,Y.C., V.F., and C.O.R. performed research; S.A.G. and J.M.H. analyzed data; A.V.S. and J.M.H.contributed new reagents/analytic tools; and S.A.G., E.B.R., C.O.R., A.V.S., and J.M.H. wrotethe paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.

See Commentary on page 2695.1To whom correspondence may be addressed. E-mail: andrew.schally@va.gov or jhare@med.miami.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1220185110/-/DCSupplemental.

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tubes than those derived from WT mice (Fig. 1 A–C). To testwhether this impairment was NO-mediated, we inhibited NOproduction in GSNOR−/− MSCs with the NO synthase inhibitorL-NG-nitroarginine methyl ester (L-NAME). This treatmentcompletely normalized the number (49.9 ± 14.8, P < 0.001) andlength (82.8 ± 20.5 μm, P < 0.001) of GSNOR−/− tubes (Fig. 1A–C), indicating that inhibition of NO production restores net-work formation by MSCs. L-NAME had no effect on WT MSCtube formation; however, treatment with the NO donors,S-nitrosoglutathione (GSNO) and S-nitroso-N-acetyl-D,L-penicil-lamine (SNAP), impaired tube formation (15.6 ± 9.0 and 31.8 ±9.9, P < 0.001) and decreased tube length (98.4 ± 56.6 and 57.1 ±24.9 μm, P < 0.001) by WTMSCs, confirming that small molecularweight S-nitrosothiols inhibit the vasculogenic potential of MSCs(Fig. S3 A–C).

MSCs from NOS2−/− Mice Produce Enhanced Capillary Tube–LikeFormation in Vitro. We also tested the impact of reducing in-tracellular NO production by assessing the vasculogenic potentialof MSCs isolated from mice lacking NOS2 (NOS2−/−). Signifi-cantly, MSCs from NOS2−/− mice formed more tubes (92.8 ± 26.0vs. 50.0 ± 15.4, P < 0.001) than WT MSCs, and the NOS2−/−

tubes were longer (121.0 ± 52.0 vs. 62.8 ± 34.6 μm, P < 0.001)than tubes from GSNOR−/− MSCs but were similar (121.0 ± 52.0vs. 124.2 ± 76.0 μm) to WT tubes (Fig. S4 A–C), further con-firming the inhibitory role of NO on MSC vasculogenesis. Wewere unable to generate MSCs from NOS1−/− mice, suggestingan indispensable role for this enzyme in MSC biology.

NO/GSNOR Modulates VEGF-A/PDGFR Signaling. Accordingly we ex-amined the expression of VEGF-A, VEGFR2, PDGFRα, andPDGFRβ in MSCs (Fig. 2 A–J). Neither WT nor GSNOR−/−MSCs expressed VEGFR2, although its ligand VEGF-A was pro-duced at similar levels by both strains (Fig. 2 A–D). PDGFRαexpression was diminished by ∼50% in GSNOR−/− MSCs as mea-sured by FACS (Fig. 2 E–G), qRT-PCR (Fig. 2H), and Westernblotting (Fig. 2 I and J). The expression of PDGFRβ did not change(Fig. 2H). Under physiologic conditions, MSCs express high levelsof PDGFRα but not VEGFR2 (16). Differentiation of MSC intoendothelial cells requires activation of the PDGFRα by VEGF-Afollowing a switch of receptors where VEGFR2 increases andPDGFRα decreases (19). Together these findings support theparadigm that environments rich in bioavailable NO or geneticmodifications (deletion of GSNOR) inhibit PDGFRα expres-sion by MSCs resulting in impaired endothelial differentiation

(Fig. 2K). Furthermore, incubation with GSNO down-regu-lated PDGFRα by approximately twofold and up-regulatedVEGF-A by∼2.5-fold inWTMSCs, supporting the actions of low-molecular-weight thiols in mediating this phenotype (Fig. S5A).Finally, in GSNOR−/− MSCs, inhibition of NOS with L-NAMEaugmented VEGF-A production ∼4.5-fold (Fig. 3A), demon-strating that NO levels modulate VEGF-A production. Activa-tion of the GHRH receptor, with JI-38, a synthetic agonist, alsoincreased VEGF-A production by 2.8-fold in GSNOR−/− MSCs(Fig. 3B).

VEGF-A/PDGFR Signaling Is a Key in MSC-Mediated Vasculogenesis.We next examined whether inhibition of PDGFRα, using a spe-cific PDGFR IV antagonist (PIV), could reduce tube formationby MSCs. Treatment with PIV (0.1 μmol/L) impaired tube for-mation in both WT (∼2.6-fold; Fig. 3 C and D; Fig. S6A) andGSNOR−/− MSCs (∼4.1 fold; Fig. 3 E and F; Fig. S6B). How-ever, the impact of PIV was much greater on GSNOR−/− tubesthan WT. In addition, while treatment with L-NAME rescuedtube formation by GSNOR−/− MSCs, PIV counteracted this ef-fect, confirming that PDGFRα activation is required for tubeformation by MSCs (Fig. 3 E and F; Fig. S6B). Furthermore,when GSNOR−/− MSCs were treated with L-NAME, VEGF-Aexpression was significantly increased (Fig. 3A), leading to en-hanced tube formation through PDGFRα activation (Fig. 3 Eand F), although mRNA expression of PDGFRα or β did notchange following NOS inhibition.

Activation of VEGF-A Production with GHRH Agonists. As an alter-native to NOS inhibition, we sought to augment VEGF-A pro-duction by activating the growth hormone–releasing hormone(GHRH) receptor, which was shown to be present on MSCs (Fig.S7 A–C). Stimulation of the GHRH receptor with the syntheticgrowth hormone–releasing hormone GHRH agonist JI-38 (20) orother potent GHRH agonists, MR-409 and MR-356 (Fig. S7 D–F), also normalized the impaired tube formation by GSNOR−/−

MSCs and did so to a similar extent as L-NAME (69 ± 11 tubesand 73 ± 33-μm tube length, P < 0.001; Fig. 3). Treatment withJI-38 and L-NAME in WT MSCs did not affect tube formation(Fig. 3 C and D; Fig. S3 A–C), although stimulation of WT MSCswith JI-38 up-regulated VEGF-A expression by ∼2.5 fold (Fig.S5B). As with L-NAME, blockade of PDGFRα abolished theimpact of this GHRH agonist (Fig. 3 E and F).

Fig. 1. Impaired capillary tube–like formation from GSNOR−/− MSCs in vitro. (A) Representative images of tube-like formation by WT and GSNOR−/− MSCsplated on Matrigel-coated plates for 0, 6, and 24 h in the presence of vehicle (rows 1 and 3) or 15 μmol/L L-NAME (rows 2 and 4) (magnification: 10×).Quantification of the number (B) and length (μm) (C) of tubes at 24 h (n ≥ 3, *P < 0.001 vs. WT, †P < 0.001 vs. GSNOR−/−).

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NO Signaling Impaired Vasculogenesis in Vivo. To confirm our invitro data, we examined vasculogenesis in vivo by performing aMatrigel plug assay. GFP-labeledMSCs fromWT andGSNOR−/−

mice were injected s.c. into immunocompromised mice (NOD-SCID). Two weeks later, Matrigel plugs were harvested andassayed for immunofluorescence staining and determination ofcapillary formation (Fig. 4A). MSCs from GSNOR−/− mice hadimpaired blood vessel formation compared withWTMSCs (1.84±1.3 vs. 10.2 ± 2.7/mm2, P < 0.01; Fig. 4 A and B). Furthermore, wedetermined the percentage of GFP-transduced MSCs that differ-entiated into endothelial cells and also formed blood vessels (Fig.4 C and D). GSNOR−/− MSCs exhibited diminished endothelialdifferentiation (7.7 ± 1.64% vs. 12.5 ± 1.0%, P < 0.02) as assessedby isolectin staining (in orange) colocalized with GFP and alsoreduced the number of GFP+ blood vessels (0.4 ± 0.2 vs. 1.6 ± 0.4mm2) compared with WT MSCs . These results agree with our invitro data and support the negative effect of NO signaling on vas-culogenesis by MSCs.

NO Donors Impaired the Ability of Human Bone Marrow–DerivedMSCs to Form Capillary Tube–Like Structures in Vitro. We nexttested whether human and mouse MSCs responded similarly toNO donors and observed that NO donors also reduced tubeformation by hMSCs. Whereas the human cells formed a net-work more rapidly than murine cells (Fig. S8), hMSCs treatedwith NO donors GSNO or SNAP produced half as many tubes asvehicle-treated cells. Treatment with 10, 40, and 100 μmol/L

GSNO reduced the number (26.6 ± 7.1, 24.8 ± 4.3, and 27.6 ±4.0, respectively, vs. 49.2.0 ± 8.1, P < 0.001) and length (94.0 ±34.3, 78.0 ± 41.0 and 85.0 ± 34.4, respectively, vs. 125.0 ± 52.6,P < 0.001) of tubes (Fig. 5 A and B). Similarly, treatment with 10and 100 μmol/L SNAP reduced tube number (34.0 ± 6.1 and 25.0 ±4.9 vs. 49.2 ± 8.1, P < 0.001) and length (82.0 ± 29.2 and 91.0 ±32.2 vs. 128 ± 52 μm, P < 0.001) compared with untreated hMSCs.Similar to murine MSCs, inhibition of NOS with L-NAME had noeffect on hMSC tube formation (Fig. 5 A and B).Finally, to test whetherMSCs respond differently to endothelial

cells, we used the Matrigel assay with human umbilical vein en-dothelial cells (HUVECs), and in contrast to mouse and humanMSCs, HUVECs treated with NO donors (GSNO and SNAP)exhibited enhanced network formation, but NOS inhibition with15 μmol/L L-NAMEwas not sufficient to reduce tube-like capillarystructure formation (Fig. S9 A–C). These experiments revealedthat NO signaling has a negative impact on endothelial differen-tiation byMSCs in contrast to (human) endothelial cells whereNOfavors angiogenesis.

DiscussionThe major finding shown in this work is that GSNOR signalingcontributes to MSC-mediated vasculogenesis. We demonstratedthis principle in a variety of ways. First, MSCs from GSNOR-de-ficient mice exhibited attenuated vasculogenesis both in vitro andin vivo. Similarly, S-nitrosothiol (SNO) donors diminished vascu-logenesis in human MSCs. Pharmacological inhibition of NO in

Fig. 2. Down-regulation of PDGFRα in GSNOR−/− MSCs. (A–C) Representative FACS analysis depicting absence of VEGFR2 (

GSNOR−/− MSCs, or genetic reduction of NO production in theNOS2−/−, enhanced vasculogenesis by MSCs. Importantly, theopposite effect was shown to be true for HUVECs in which NO

enhanced vascular tube formation. Together, these findings reveala unique, paradoxical mode of vascular regulation between MSCsand endothelial cells and suggest that MSC-mediated vascularformation may increase in states of NO deficiency. Importantly,the simultaneous potentiation of EPC- and inhibition of MSC-mediated vasculogenesis by NO may represent a mechanism forpreserving MSC regulatory capacity in an environment of en-hanced EPC vascular formation.NO signaling in MSCs has heretofore not been examined. The

present study revealed that GSNOR deficiency impairs MSC-mediated postnatal vasculogenesis. Previously, we and othersshowed that GSNOR is a key regulator of cardiovascular func-tion and vascular tone, regulating a dynamic nitrosylation/deni-trosylation cycle of proteins (21, 22). We chose to investigateS-nitrosylation as the primary signaling mode exerting NO bio-activity and took advantage of the fact that both excessive NOproduction by NOS activation or reduced SNO metabolism dueto GSNOR deficiency enhances S-nitrosylation (23). Accord-ingly, we used MSCs from the GSNOR−/− as the primary modeof investigating NO/SNO signaling in these stem cells.At the mechanistic level, our studies reveal that the underlying

basis by whichNO signaling throughGSNORdirectly affectsMSCvasculogenesis appears to be regulation of PDGFRα abundance(Fig. 2K). Bone marrow MSCs, which do not express VEGFR2(16), respond to VEGF-A through a PDGFR–ligand interaction(16, 24, 25). Here we show that GSNOR−/− MSCs have reducedexpression of PDGFRα, which is causally linked to impaired en-dothelial differentiation. Importantly, treatment with L-NAMErescued capillary network formation from GSNOR−/− MSCs byincreasing VEGF-A production followed by activation ofPDGFRα. However, this effect was not observed in WT MSCswhen similarly treated with L-NAME, which may represent celltype differences in dose response. Human MSCs cocultured withendothelial cells differentiate into endothelial-like cells, and thisprocess can be inhibited by VEGF-A antisera (26), corroboratingour finding that VEGF-A is crucial for MSC-mediated vasculo-genesis. In our study, we demonstrated clearly that VEGF-A/PDGFRα directly affected MSC fate decisions, although we can-not discount that the interactions between MSCs and endothelialcells are crucial for vascular homeostasis and repair. In this re-spect, MSCs are thought to be intimately involved with vascularhomeostasis throughout the body, preserving vascular integrity bydifferentiating to pericytes (9, 27).

Fig. 3. VEGF-A/PDGFR signaling in MSC-mediated vasculogenesis. (A and B)GSNORMSCs increase VEGF-A production when stimulated by L-NAME and JI-38. Quantification of tube formation on Matrigel shows that MSCs from bothWT (C and D) and GSNOR−/− (E and F) mice exhibit reduced tube number andlength (μm) when treated with PDGFRα inhibitor (PIV) either alone or in thepresence of 0.25 μmol/L JI-38 or 15 μmol/L L-NAME. (n ≥ 3, *P < 0.05).

Fig. 4. MSCs from GSNOR−/− mice exhibit reduced endothelial differentiation and impaired blood vessel formation in vivo. (A and B) Matrigel plug (2 wkafter injection) containing GFP+ MSCs from WT and GSNOR−/−. (B) H&E staining with blood vessel formation indicated by the black arrows (C) MSCs fromGSNOR−/− mice following isolectin (red) and GFP (green) staining shows colocalization (orange) and exhibit diminished potential to differentiate into en-dothelial cells than WT MSCs. (D) Quantification of the number of GFP+ blood vessels containing autofluorescent red blood cells [white arrows, see (A)]. (E)Quantification of blood vessel formation 2 wk after injection. *P < 0.05.

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We also used a second strategy to test whether VEGF-A sig-naling rescued diminished vasculogenesis by GSNOR−/− MSCs.Based on our previous observations, that the activation of GHRHreceptors leads to an increased VEGF-A in vivo (28), we usedGHRH agonists. Indeed these agonists normalized vasculogenesisin a PDGFRα-dependent manner. These findings have importantimplications for the therapeutic application of GHRH agonists toischemic disorders. We previously showed that GHRH agonistsimprove cardiac structure and function after myocardial infarction(28, 29). These findings shed further light on the beneficial effectsof GHRH agonists on wound healing (30) and the maintenance ofpancreatic β islets (31).Our results confirmed that NO signaling through small-mo-

lecular-weight thiols is an important regulator of vasculogenesisin MSCs. More specifically, we found that endothelial differen-tiation is impaired in GSNOR−/− MSCs in vivo, suggesting thatGSNOR represents a valuable pharmacologic target for regu-lation of neovascularization. Moreover, our results support thatS-nitrosylation plays an important role in MSC-mediated vas-culogenesis and may affect cell fate decisions. Importantly, Limaet al. (22) showed that myocardial infarction size is reduced inGSNOR−/− mice, an effect that was associated with enhancedneovascularization, suggesting that neovascularization in thatsetting is primarily a result of EPC activation.In addition, we demonstrated that decreased production of NO

enhanced tube-like formation on Matrigel in vitro. This result wasillustrated by the increased tube formation by MSCs fromNOS2−/− mice, the normalization of tube formation by treatmentof GSNOR−/− MSCs with L-NAME, and ultimately, the re-duction in tube formation in WT mMSCs and in hMSCs trea-ted with NO donors, indicating a cross-species effect.Therefore, deficiency of NO has a positive impact on MSC-

mediated vasculogenesis. In agreement with our data, Wanget al. (32) demonstrated that treatment of human MSCs withNO donors suppresses production of the proangiogenic factorsVEGF and hepatocyte growth factor. Moreover, bone marrow–derived MSCs isolated from patients with systemic sclerosis,a disease characterized by NO overproduction, vascular dys-function, and systemic fibrosis, have impaired endothelial celldifferentiation (33), consistent with our findings that environ-ments rich in NO impair postnatal vasculogenesis by MSCs.Together, these findings suggest a reduced regenerative capacityof MSCs in nitroso/redox unbalanced environments such as

heart failure, sepsis, and neuronal degenerative diseases (34).Furthermore, NO may attenuate the protection by MSCs in is-chemic myocardium by serving as a natural braking mechanismfor MSC-induced neovascularization in a tissue recovering fromischemic injury.We showed that NO synthases, NOS1 and NOS2, but not

NOS3, were constitutively expressed in MSCs. Importantly,MSCs from WT and GSNOR−/− mice had equivalent NOproduction; however, NOS1 expression was up-regulated inGSNOR−/− MSCs, presumably to maintain NO production atphysiologic levels. Unlike MSCs, endothelial cells express theNOS3 isoform (35) shown to participate in proangiogenic sig-naling (36). Additionally, NOS3 and GSNOR play an importantrole in endothelial cell–mediated postnatal angiogenesis andvascular tone (21, 22). Moreover, GSNOR−/− mice have aug-mented myocardial capillary density, at baseline, as shown byCD31 staining (22), suggesting that S-nitrosylation enhances an-giogenesis by endothelial cells in contrast to the inhibitory effect onMSCs. We observed a similar effect in murine and human MSCscompared with endothelial cells (HUVECs), suggesting that NOpromotes angiogenesis by endothelial cells and inhibits vasculo-genesis by MSC. Moreover, pathological conditions such as endo-thelial dysfunction where there is reduced NO bioavailability (37)may trigger vasculogenesis by MSC, perhaps serving as a compen-satory mechanism.In summary, our findings offer insight regarding the role of

NO in vascular biology in which environments that are NO de-ficient trigger the participation of MSCs in vasculogenesis. Thus,NO exerts a balanced effect on the different cellular precursorsparticipating in neo-angiogenesis, promoting that portion me-diated by EPCs while simultaneously inhibiting that originatingwith MSC-like cells. These findings offer exciting insights intothe pathophysiology of conditions characterized by exuberantneo-vascularization such as cancer and diabetic retinopathy and,as such, have therapeutic implications.

Materials and MethodsA detailed description of the materials and methods can be found in SIMaterials and Methods. Briefly, GSNOR−/− mice were generated as described(18) and compared with age- and sex-matched NOS2−/− and WT mice (C57BL/6). Bone marrow–derived MSCs isolated and expanded from WT, GSNOR−/−,and NOS2−/− mice and humans (hMSCs) were grown in endothelial growthmedia (EGM-2; Lonza) followed by 24 h in Matrigel, in the presence of ve-hicle, L-NAME [an NO synthase (NOS) inhibitor], GSNO, and SNAP (NOdonors), and JI-38 (20), a GHRH agonist. GHRH agonists, JI-38, MR-409, andMR-356 were synthesized in the laboratory of A.V.S. NO production and NOSexpression by MSCs was assessed. Additionally, we used an allograft assay tostudy in vivo vasculogenesis by murine MSCs. All animal protocols and ex-perimental procedures were approved by the University of Miami In-stitutional Animal Care and Use Committee.

Data were analyzed for significance using one-way ANOVA, the Tukey-Kramer multiple comparisons test, and Student t test. All analyses wereperformed using GraphPad Prism, version 4.03, and P < 0.05 was consideredsignificant. All data were presented as mean ± SE.

ACKNOWLEDGMENTS. We thank Irene Margitich, Lauro M. Takeuchi, andMirella Figueroa for technical assistance; Carmen Perez for preparing thehistologic sections; Shannon Opiela, Jay Enten, and James Phillips for FACSanalysis; and Dr. Norman Block for editorial suggestions. This work wasfunded by National Heart, Lung, and Blood Institute Grants R01 HL-094849,R01 HL084275, RO1 HL107110, and R01 HL110737 (to J.M.H.) and a W. H.Coulter Center Award [Medical Research Service of Veterans Affairs andDepartments of Pathology and Medicine, Division of Hematology/Oncology,University of Miami Miller School of Medicine and South Florida VeteransAffairs Foundation for Research and Education (to A.V.S.)].

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