(GSNOR) Enhances Vasculogenesis by Mesenchymal Stem Cells

S-nitrosoglutathione reductase (GSNOR) enhances vasculogenesis by mesenchymal stem cells

Samirah 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 and differentiation of EPCs and MSCs, we reasoned that NO plays maturation of endothelial progenitor cells, its role in the differen- an equivalent role in this process. tiation of mesenchymal stem cells (MSCs) into endothelial cells Accordingly, we tested the hypothesis that NO signaling, me- remains controversial. We tested the role of NO signaling in MSCs diated by small molecular weight thiols (molecular weight < 500), derived from WT mice and mice homozygous for a deletion of promotes MSC differentiation into endothelial cells. To test our − − S-nitrosoglutathione reductase (GSNOR / ), a denitrosylase that hypothesis, we assessed the functional consequences of deletion − − regulates S-nitrosylation. GSNOR / MSCs exhibited markedly of S-nitrosoglutathione reductase (GSNOR), which in turn diminished capacity for vasculogenesis in an in vitro Matrigel increases S-nitrosothiols (17, 18), on MSC-mediated vasculo- − − tube–forming assay and in vivo relative to WT MSCs. This decrease genesis. We report that paradoxically, MSCs from GSNOR / was associated with down-regulation of the PDGF receptorα mice exhibit diminished endothelial differentiation, thereby − − (PDGFRα) in GSNOR / MSCs, a receptor essential for VEGF-A demonstrating an inhibitory effect of S-nitrosylation on vascu- action in MSCs. Pharmacologic inhibition of NO synthase with logenesis mediated by MSCs. G L-N -nitroarginine methyl ester (L-NAME) and stimulation of growth hormone–releasing hormone receptor (GHRHR) with GHRH agonists Results −/− augmented VEGF-A production and normalized tube formation in Murine Bone Marrow MSC Characterization. Both WT and GSNOR - −/− GSNOR MSCs, whereas NO donors or PDGFR antagonist re- derived MSCs were spindle shaped, adherent to plastic tissue duced tube formation ∼50% by murine and human MSCs. The culture dishes (Fig. S1A), and negative for Lineage, a mixture of antagonist also blocked the rescue of tube formation in hematopoietic markers, CD34 and CD45, and positive for stem −/− GSNOR MSCs by L-NAME or the GHRH agonists JI-38, MR-409, cell antigens SCA-1, CD73, CD90.2, and CD105 (Fig. S1 B and C). and MR-356. Therefore, GSNOR−/− MSCs have a deficient capacity for endothelial differentiation due to downregulation of PDGFRα NO Signaling and MSCs. Denitrosylation is controlled in significant related to NO/GSNOR imbalance. These findings unravel important part by GSNOR, a unique and specific enzyme for which S-nitro- aspects of modulation of MSCs by VEGF-A activation of the PDGFR soglutathione (GSNO) is the substrate (18) (Fig. S2A). Neither −/− and illustrate a paradoxical inhibitory role of S-nitrosylation sig- MSCs (Fig. S2 B and C) nor liver (Fig. S2D)fromGSNOR mice naling in MSC vasculogenesis. Accordingly, disease states charac- expresses GSNOR. NOS1 and NOS2 were constitutively expressed terized by NO deficiency may trigger MSC-mediated vasculogenesis. in both strains of MSCs (Fig. S2 E and F), whereas NOS3 expres- fi These ndings have important implications for therapeutic applica- sion was absent from both strains (Fig. S2G). Interestingly, NOS1 −/− tion of GHRH agonists to ischemic disorders. mRNA was ∼100-fold higher in GSNOR mice (2.7 × 103 ± 3 × 102 absolute number of transcripts, ΔCt) compared with WT MSCs – angiogenesis | nitroso redox imbalance (2 × 104 ± 2 × 103, P < 0.05; Fig. S2E). Despite the up-regulation of NOS1, actual NO production, measured by 4,5-diamino-fluores- itric oxide (NO) and VEGF signaling promotes vasculogenesis cein diacetate (DAF-2DA), was nearly identical between the Nby endothelial progenitor cells (EPCs) (1–5). For example, strains (Fig. S2H). Thus, GSNOR deficiency up-regulates NOS1 −/− mice deficient in endothelial NO synthase (NOS3 ) show re- but does not lead to increased NO production. duced VEGF-induced mobilization of bone marrow progenitor − − cells to sites of injury (4). In EPC-mediated vasculogenesis, VEGF-A GSNOR / MSCs Exhibit Impaired Formation of Capillary Tube–Like activates its major receptor [VEGF receptor 2 (VEGFR2)], trig- Structures in Vitro. Next we used a Matrigel assay to analyze the gering cell differentiation into mature endothelial cells (ECs) and ability of MSCs to form tube-like structures in vitro. Cells were grown in endothelial medium (EGM-2; Lonza) for 1 wk before enhancing angiogenesis (6, 7). −/− Mesenchymal stem cells (MSCs) also participate in postnatal plating on Matrigel. Surprisingly, GSNOR -derived MSCs angiogenesis (8, 9), and vascular pericytes, which are crucial for formed significantly fewer (29.9 ± 11.75 vs. 50.2 ± 14.04, P < maintaining vascular integrity, share similar phenotypic features 0.001) and shorter (62.8 ± 14.0 vs. 124.2 ± 49.0 μm, P < 0.001) with MSCs (10). Exogenously administered, MSCs readily form new capillaries and medium-sized arteries (11, 12), properties important for the tissue regenerative capacity of MSCs (13). We 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. and others have shown that MSCs differentiate into endothelial contributed new reagents/analytic tools; and S.A.G., E.B.R., C.O.R., A.V.S., and J.M.H. wrote cells in vitro (14) and in vivo and contribute to neovascularization, the paper. particularly during tissue ischemia and tumor vascularization (8, The authors declare no conflict of interest. 11, 12, 15). As with EPCs, VEGF also plays an important role in Freely available online through the PNAS open access option. stimulating MSC differentiation, but does so by activating the See Commentary on page 2695. PDGF receptor (PDGFR) as opposed to the VEGFR2, which is 1To whom correspondence may be addressed. E-mail: [email protected] or jhare@ absent on MSCs (16). However, the impact of NO signaling in the med.miami.edu. differentiation of MSCs into endothelial cells has not been pre- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. viously tested. Given the similar signaling involved in endothelial 1073/pnas.1220185110/-/DCSupplemental.

2834–2839 | PNAS | February 19, 2013 | vol. 110 | no. 8 www.pnas.org/cgi/doi/10.1073/pnas.1220185110 Downloaded by guest on September 27, 2021 SEE COMMENTARY

− − − − Fig. 1. Impaired capillary tube–like formation from GSNOR / MSCs in vitro. (A) Representative images of tube-like formation by WT and GSNOR / MSCs plated 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−/−).

tubes than those derived from WT mice (Fig. 1 A–C). To test (Fig. 2K). Furthermore, incubation with GSNO down-regu- whether this impairment was NO-mediated, we inhibited NO lated PDGFRα by approximately twofold and up-regulated −/− production in GSNOR MSCs with the NO synthase inhibitor VEGF-A by ∼2.5-fold in WT MSCs, supporting the actions of low- G L-N -nitroarginine methyl ester (L-NAME). This treatment molecular-weight thiols in mediating this phenotype (Fig. S5A). completely normalized the number (49.9 ± 14.8, P < 0.001) and −/− Finally, in GSNOR MSCs, inhibition of NOS with L-NAME CELL BIOLOGY ± μ P < −/− length (82.8 20.5 m, 0.001) of GSNOR tubes (Fig. 1 augmented VEGF-A production ∼4.5-fold (Fig. 3A), demon- A–C ), indicating that inhibition of NO production restores net- strating that NO levels modulate VEGF-A production. Activa- work formation by MSCs. L-NAME had no effect on WT MSC tion of the GHRH receptor, with JI-38, a synthetic agonist, also − − tube formation; however, treatment with the NO donors, increased VEGF-A production by 2.8-fold in GSNOR / MSCs S-nitrosoglutathione (GSNO) and S-nitroso-N-acetyl-D,L-penicil- (Fig. 3B). lamine (SNAP), impaired tube formation (15.6 ± 9.0 and 31.8 ± P < ± ± 9.9, 0.001) and decreased tube length (98.4 56.6 and 57.1 VEGF-A/PDGFR Signaling Is a Key in MSC-Mediated Vasculogenesis. 24.9 μm, P < 0.001) by WT MSCs, confirming that small molecular α S We next examined whether inhibition of PDGFR , using a spe- weight -nitrosothiols inhibit the vasculogenic potential of MSCs cific PDGFR IV antagonist (PIV), could reduce tube formation (Fig. S3 A–C). by MSCs. Treatment with PIV (0.1 μmol/L) impaired tube for- − − ∼ C D A MSCs from NOS2 / Mice Produce Enhanced Capillary Tube–Like mation in both WT ( 2.6-fold; Fig. 3 and ; Fig. S6 ) and −/− ∼ E F B Formation in Vitro. We also tested the impact of reducing in- GSNOR MSCs ( 4.1 fold; Fig. 3 and ; Fig. S6 ). How- −/− tracellular NO production by assessing the vasculogenic potential ever, the impact of PIV was much greater on GSNOR tubes − − of MSCs isolated from mice lacking NOS2 (NOS2 / ). Signifi- than WT. In addition, while treatment with L-NAME rescued − − − − cantly, MSCs from NOS2 / mice formed more tubes (92.8 ± 26.0 tube formation by GSNOR / MSCs, PIV counteracted this ef- −/− vs. 50.0 ± 15.4, P < 0.001) than WT MSCs, and the NOS2 fect, confirming that PDGFRα activation is required for tube tubes were longer (121.0 ± 52.0 vs. 62.8 ± 34.6 μm, P < 0.001) formation by MSCs (Fig. 3 E and F; Fig. S6B). Furthermore, −/− −/− than tubes from GSNOR MSCs but were similar (121.0 ± 52.0 when GSNOR MSCs were treated with L-NAME, VEGF-A vs. 124.2 ± 76.0 μm) to WT tubes (Fig. S4 A–C), further con- expression was significantly increased (Fig. 3A), leading to en- firming the inhibitory role of NO on MSC vasculogenesis. We α E −/− hanced tube formation through PDGFR activation (Fig. 3 were unable to generate MSCs from NOS1 mice, suggesting and F), although mRNA expression of PDGFRα or β did not an indispensable role for this enzyme in MSC biology. change following NOS inhibition.

NO/GSNOR Modulates VEGF-A/PDGFR Signaling. Accordingly we ex- Activation of VEGF-A Production with GHRH Agonists. As an alter- α amined the expression of VEGF-A, VEGFR2, PDGFR ,and native to NOS inhibition, we sought to augment VEGF-A pro- β A–J −/− PDGFR in MSCs (Fig. 2 ). Neither WT nor GSNOR duction by activating the growth hormone–releasing hormone MSCs expressed VEGFR2, although its ligand VEGF-A was pro- (GHRH) receptor, which was shown to be present on MSCs (Fig. duced at similar levels by both strains (Fig. 2 A–D). PDGFRα − − S7 A–C). Stimulation of the GHRH receptor with the synthetic expression was diminished by ∼50% in GSNOR / MSCs as mea- growth hormone–releasing hormone GHRH agonist JI-38 (20) or sured by FACS (Fig. 2 E–G), qRT-PCR (Fig. 2H), and Western I J β other potent GHRH agonists, MR-409 and MR-356 (Fig. S7 D– blotting (Fig. 2 and ). The expression of PDGFR did not change F −/− (Fig. 2H). Under physiologic conditions, MSCs express high levels ), also normalized the impaired tube formation by GSNOR ± of PDGFRα but not VEGFR2 (16). Differentiation of MSC into MSCs and did so to a similar extent as L-NAME (69 11 tubes ± μ P < endothelial cells requires activation of the PDGFRα by VEGF-A and 73 33- m tube length, 0.001; Fig. 3). Treatment with following a switch of receptors where VEGFR2 increases and JI-38 and L-NAME in WT MSCs did not affect tube formation PDGFRα decreases (19). Together these ﬁndings support the (Fig. 3 C and D; Fig. S3 A–C), although stimulation of WT MSCs paradigm that environments rich in bioavailable NO or genetic with JI-38 up-regulated VEGF-A expression by ∼2.5 fold (Fig. modiﬁcations (deletion of GSNOR) inhibit PDGFRα expres- S5B). As with L-NAME, blockade of PDGFRα abolished the sion by MSCs resulting in impaired endothelial differentiation impact of this GHRH agonist (Fig. 3 E and F).

Gomes et al. PNAS | February 19, 2013 | vol. 110 | no. 8 | 2835 Downloaded by guest on September 27, 2021 − − − − Fig. 2. Down-regulation of PDGFRα in GSNOR / MSCs. (A–C) Representative FACS analysis depicting absence of VEGFR2 (<1%) in WT and GSNOR / MSCs. (D) Both strains produce VEGF-A. (E–G) PDGFRα expression is reduced in GSNOR−/− MSCs compared with WT. (H) PDGFRα as assessed by qRT-PCR is down- regulated ∼1.8-fold in GSNOR−/− MSCs. (I and J) Western blot and quantitation of band density for PDGFRα (n ≥ 3, *P < 0.05). (K) Model of MSC differentiation toward endothelial cells.

NO Signaling Impaired Vasculogenesis in Vivo. To confirm our in GSNO reduced the number (26.6 ± 7.1, 24.8 ± 4.3, and 27.6 ± vitro data, we examined vasculogenesis in vivo by performing a 4.0, respectively, vs. 49.2.0 ± 8.1, P < 0.001) and length (94.0 ± −/− Matrigel plug assay. GFP-labeled MSCs from WT and GSNOR 34.3, 78.0 ± 41.0 and 85.0 ± 34.4, respectively, vs. 125.0 ± 52.6, mice were injected s.c. into immunocompromised mice (NOD- P < 0.001) of tubes (Fig. 5 A and B). Similarly, treatment with 10 SCID). Two weeks later, Matrigel plugs were harvested and and 100 μmol/L SNAP reduced tube number (34.0 ± 6.1 and 25.0 ± assayed for immunofluorescence staining and determination of 4.9 vs. 49.2 ± 8.1, P < 0.001) and length (82.0 ± 29.2 and 91.0 ± −/− capillary formation (Fig. 4A). MSCs from GSNOR mice had 32.2 vs. 128 ± 52 μm, P < 0.001) compared with untreated hMSCs. impaired blood vessel formation compared with WT MSCs (1.84 ± Similar to murine MSCs, inhibition of NOS with L-NAME had no 1.3 vs. 10.2 ± 2.7/mm2, P < 0.01; Fig. 4 A and B). Furthermore, we effect on hMSC tube formation (Fig. 5 A and B). determined the percentage of GFP-transduced MSCs that differ- Finally, to test whether MSCs respond differently to endothelial entiated into endothelial cells and also formed blood vessels (Fig. cells, we used the Matrigel assay with human umbilical vein en- −/− 4 C and D). GSNOR MSCs exhibited diminished endothelial dothelial cells (HUVECs), and in contrast to mouse and human differentiation (7.7 ± 1.64% vs. 12.5 ± 1.0%, P < 0.02) as assessed MSCs, HUVECs treated with NO donors (GSNO and SNAP) by isolectin staining (in orange) colocalized with GFP and also exhibited enhanced network formation, but NOS inhibition with + reduced the number of GFP blood vessels (0.4 ± 0.2 vs. 1.6 ± 0.4 15 μmol/L L-NAME was not sufficient to reduce tube-like capillary mm2) compared with WT MSCs . These results agree with our in structure formation (Fig. S9 A–C). These experiments revealed vitro data and support the negative effect of NO signaling on vas- that NO signaling has a negative impact on endothelial differen- culogenesis by MSCs. tiation by MSCs in contrast to (human) endothelial cells where NO favors angiogenesis. NO Donors Impaired the Ability of Human Bone Marrow–Derived MSCs to Form Capillary Tube–Like Structures in Vitro. We next Discussion tested whether human and mouse MSCs responded similarly to The major finding shown in this work is that GSNOR signaling NO donors and observed that NO donors also reduced tube contributes to MSC-mediated vasculogenesis. We demonstrated formation by hMSCs. Whereas the human cells formed a net- this principle in a variety of ways. First, MSCs from GSNOR-de- work more rapidly than murine cells (Fig. S8), hMSCs treated ficient mice exhibited attenuated vasculogenesis both in vitro and with NO donors GSNO or SNAP produced half as many tubes as in vivo. Similarly, S-nitrosothiol (SNO) donors diminished vascu- vehicle-treated cells. Treatment with 10, 40, and 100 μmol/L logenesis in human MSCs. Pharmacological inhibition of NO in

2836 | www.pnas.org/cgi/doi/10.1073/pnas.1220185110 Gomes et al. Downloaded by guest on September 27, 2021 enhanced vascular tube formation. Together, these findings reveal a unique, paradoxical mode of vascular regulation between MSCs SEE COMMENTARY and endothelial cells and suggest that MSC-mediated vascular formation 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 for preserving MSC regulatory capacity in an environment of enhanced 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 others showed that GSNOR is a key regulator of cardiovascular function and vascular tone, regulating a dynamic nitrosylation/denitrosylation cycle of proteins (21, 22). We chose to investigate S-nitrosylation as the primary signaling mode exerting NO bio- activity and took advantage of the fact that both excessive NO production by NOS activation or reduced SNO metabolism due to GSNOR deficiency enhances S-nitrosylation (23). Accord- −/− ingly, we used MSCs from the GSNOR as the primary mode of investigating NO/SNO signaling in these stem cells. At the mechanistic level, our studies reveal that the underlying basis by which NO signaling through GSNOR directly affects MSC vasculogenesis 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 reduced expression of PDGFRα, which is causally linked to impaired endothelial differentiation. Importantly, treatment with L-NAME CELL BIOLOGY −/− rescued capillary network formation from GSNOR MSCs by increasing VEGF-A production followed by activation of PDGFRα. However, this effect was not observed in WT MSCs when similarly treated with L-NAME, which may represent cell type differences in dose response. Human MSCs cocultured with Fig. 3. VEGF-A/PDGFR signaling in MSC-mediated vasculogenesis. (A and B) endothelial cells differentiate into endothelial-like cells, and this GSNOR MSCs increase VEGF-A production when stimulated by L-NAME and JI- process can be inhibited by VEGF-A antisera (26), corroborating 38. Quantification of tube formation on Matrigel shows that MSCs from both our finding that VEGF-A is crucial for MSC-mediated vasculo- −/− WT (C and D) and GSNOR (E and F) mice exhibit reduced tube number and genesis. In our study, we demonstrated clearly that VEGF-A/ μ α length ( m) when treated with PDGFR inhibitor (PIV) either alone or in the PDGFRα directly affected MSC fate decisions, although we can- presence of 0.25 μmol/L JI-38 or 15 μmol/L L-NAME. (n ≥ 3, *P < 0.05). not discount that the interactions between MSCs and endothelial

−/− cells are crucial for vascular homeostasis and repair. In this re- GSNOR MSCs, or genetic reduction of NO production in the spect, MSCs are thought to be intimately involved with vascular − − NOS2 / , enhanced vasculogenesis by MSCs. Importantly, the homeostasis throughout the body, preserving vascular integrity by opposite effect was shown to be true for HUVECs in which NO differentiating to pericytes (9, 27).

Fig. 4. MSCs from GSNOR−/− mice exhibit reduced endothelial differentiation and impaired blood vessel formation in vivo. (A and B) Matrigel plug (2 wk after injection) containing GFP+ MSCs from WT and GSNOR−/−.(B) H&E staining with blood vessel formation indicated by the black arrows (C) MSCs from − − GSNOR / mice following isolectin (red) and GFP (green) staining shows colocalization (orange) and exhibit diminished potential to differentiate into endothelial 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.

Gomes et al. PNAS | February 19, 2013 | vol. 110 | no. 8 | 2837 Downloaded by guest on September 27, 2021 heart failure, sepsis, and neuronal degenerative diseases (34). Furthermore, NO may attenuate the protection by MSCs in ischemic myocardium by serving as a natural braking mechanism for MSC-induced neovascularization in a tissue recovering from ischemic 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 NO production; however, NOS1 expression was up-regulated in −/− GSNOR MSCs, presumably to maintain NO production at physiologic levels. Unlike MSCs, endothelial cells express the NOS3 isoform (35) shown to participate in proangiogenic signaling (36). Additionally, NOS3 and GSNOR play an important Fig. 5. Exposure of human MSCs to NO donors impairs the formation of role in endothelial cell–mediated postnatal angiogenesis and −/− capillary tube-like structures (A and B). Quantification of the (A) number and vascular tone (21, 22). Moreover, GSNOR mice have aug- (B) length (μm) of tube-like structures following 6-h exposure to no treat- mented myocardial capillary density, at baseline, as shown by ment, vehicle, L-NAME, GSNO, or SNAP (n ≥ 3, *P < 0.05 vs. hMSCS ± vehicle). CD31 staining (22), suggesting that S-nitrosylation enhances angiogenesis by endothelial cells in contrast to the inhibitory effect on MSCs. We observed a similar effect in murine and human MSCs We also used a second strategy to test whether VEGF-A sig- −/− compared with endothelial cells (HUVECs), suggesting that NO naling rescued diminished vasculogenesis by GSNOR MSCs. promotes angiogenesis by endothelial cells and inhibits vasculo- Based on our previous observations, that the activation of GHRH genesis by MSC. Moreover, pathological conditions such as endo- receptors leads to an increased VEGF-A in vivo (28), we used thelial dysfunction where there is reduced NO bioavailability (37) GHRH agonists. Indeed these agonists normalized vasculogenesis may trigger vasculogenesis by MSC, perhaps serving as a compen- α fi in a PDGFR -dependent manner. These ndings have important satory mechanism. implications for the therapeutic application of GHRH agonists to In summary, our findings offer insight regarding the role of ischemic disorders. We previously showed that GHRH agonists NO in vascular biology in which environments that are NO de- improve cardiac structure and function after myocardial infarction fi fi fi cient trigger the participation of MSCs in vasculogenesis. Thus, (28, 29). These ndings shed further light on the bene cial effects NO exerts a balanced effect on the different cellular precursors of GHRH agonists on wound healing (30) and the maintenance of participating in neo-angiogenesis, promoting that portion me- pancreatic β islets (31). fi diated by EPCs while simultaneously inhibiting that originating Our results con rmed that NO signalingthroughsmall-mo- with MSC-like cells. These findings offer exciting insights into lecular-weight thiols is an important regulator of vasculogenesis the pathophysiology of conditions characterized by exuberant in MSCs. More specifically, we found that endothelial differen- −/− neo-vascularization such as cancer and diabetic retinopathy and, tiationisimpairedinGSNOR MSCs in vivo, suggesting that as such, have therapeutic implications. GSNOR represents a valuable pharmacologic target for regulation of neovascularization. Moreover, our results support that Materials and Methods S -nitrosylation plays an important role in MSC-mediated vas- A detailed description of the materials and methods can be found in SI − − culogenesis and may affect cell fate decisions. Importantly, Lima Materials and Methods. Briefly, GSNOR / mice were generated as described et al. (22) showed that myocardial infarction size is reduced in (18) and compared with age- and sex-matched NOS2−/− and WT mice (C57BL/ −/− − − GSNOR mice, an effect that was associated with enhanced 6). Bone marrow–derived MSCs isolated and expanded from WT, GSNOR / , − − neovascularization, suggesting that neovascularization in that and NOS2 / mice and humans (hMSCs) were grown in endothelial growth setting is primarily a resultofEPCactivation. media (EGM-2; Lonza) followed by 24 h in Matrigel, in the presence of ve- In addition, we demonstrated that decreased production of NO hicle, L-NAME [an NO synthase (NOS) inhibitor], GSNO, and SNAP (NO enhanced tube-like formation on Matrigel in vitro. This result was donors), and JI-38 (20), a GHRH agonist. GHRH agonists, JI-38, MR-409, and illustrated by the increased tube formation by MSCs from MR-356 were synthesized in the laboratory of A.V.S. NO production and NOS −/− expression by MSCs was assessed. Additionally, we used an allograft assay to NOS2 mice, the normalization of tube formation by treatment −/− study in vivo vasculogenesis by murine MSCs. All animal protocols and ex- of GSNOR MSCs with L-NAME, and ultimately, the re- perimental procedures were approved by the University of Miami In- duction in tube formation in WT mMSCs and in hMSCs trea- stitutional Animal Care and Use Committee. ted with NO donors, indicating a cross-species effect. Data were analyzed for significance using one-way ANOVA, the Tukey- Therefore, deficiency of NO has a positive impact on MSC- Kramer multiple comparisons test, and Student t test. All analyses were mediated vasculogenesis. In agreement with our data, Wang performed using GraphPad Prism, version 4.03, and P < 0.05 was considered et al. (32) demonstrated that treatment of human MSCs with significant. All data were presented as mean ± SE. NO donors suppresses production of the proangiogenic factors VEGF and hepatocyte growth factor. Moreover, bone marrow– ACKNOWLEDGMENTS. We thank Irene Margitich, Lauro M. Takeuchi, and Mirella Figueroa for technical assistance; Carmen Perez for preparing the derived MSCs isolated from patients with systemic sclerosis, histologic sections; Shannon Opiela, Jay Enten, and James Phillips for FACS a disease characterized by NO overproduction, vascular dys- analysis; and Dr. Norman Block for editorial suggestions. This work was function, and systemic fibrosis, have impaired endothelial cell funded by National Heart, Lung, and Blood Institute Grants R01 HL-094849, differentiation (33), consistent with our findings that environ- R01 HL084275, RO1 HL107110, and R01 HL110737 (to J.M.H.) and a W. H. ments rich in NO impair postnatal vasculogenesis by MSCs. Coulter Center Award [Medical Research Service of Veterans Affairs and fi Departments of Pathology and Medicine, Division of Hematology/Oncology, Together, these ndings suggest a reduced regenerative capacity University of Miami Miller School of Medicine and South Florida Veterans of MSCs in nitroso/redox unbalanced environments such as Affairs Foundation for Research and Education (to A.V.S.)].

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