Proangiogenic Potential Research Articles

The neurotrophic receptor Ntrk2 directs lymphoid tissue neovascularization during Leishmania donovani infection.

The neurotrophic tyrosine kinase receptor type 2 (Ntrk2, also known as TrkB) and its ligands brain derived neurotrophic factor (Bdnf), neurotrophin-4 (NT-4/5), and neurotrophin-3 (NT-3) are known primarily for their multiple effects on neuronal differentiation and survival. Here, we provide evidence that Ntrk2 plays a role in the pathologic remodeling of the spleen that accompanies chronic infection. We show that in Leishmania donovani-infected mice, Ntrk2 is aberrantly expressed on splenic endothelial cells and that new maturing blood vessels within the white pulp are intimately associated with F4/80hiCD11bloCD11c+ macrophages that express Bdnf and NT-4/5 and have pro-angiogenic potential in vitro. Furthermore, administration of the small molecule Ntrk2 antagonist ANA-12 to infected mice significantly inhibited white pulp neovascularization but had no effect on red pulp vascular remodeling. We believe this to be the first evidence of the Ntrk2/neurotrophin pathway driving pathogen-induced vascular remodeling in lymphoid tissue. These studies highlight the therapeutic potential of modulating this pathway to inhibit pathological angiogenesis.

An increase in peak excess pressure accounts for the rise in systolic blood pressure along the aorta

Endothelial cells derived from induced pluripotent stem cells (iPSC-ECs) have been shown to promote angiogenesis in vivo. Recently, an alternative method of generating endothelial cells has been developed, by directly differentiating fibroblasts into endothelial cells (iECs), bypassing the pluripotent intermediate. Here we compared survival, engraftment and pro-angiogenic potential of iECs versus iPSC-ECs in a mouse model of hindlimb ischaemia. Human iPSC-ECs and iECs were transduced with a firefly luciferase and green fluorescent protein reporter for bioluminescence imaging. They exhibited endothelial behavior in vitro, assessed by migration towards VEGF, uptake of acetylated-LDL and tubule formation on matrigel. NODSCID mice (n=7-9) underwent unilateral femoral artery ligation and received either control vehicle or 1x106 iPSCECs or iECs, via intramuscular injection. Recovery of the ischaemic limb was tracked for 14 days. Laser Doppler imaging after femoral artery ligation revealed enhanced blood perfusion recovery in the iEC group vs. control at day 7 (p<0.05), 10 and 14 (p<0.001). IPSC-ECs enhanced recovery vs. control at day 14 (p<0.05). At day 10, perfusion was significantly greater in iEC vs. iPSC-EC treated mice (p<0.01). IPSC-EC and iEC bioluminescent signals decreased over 14 days, but remained above background at all observed timepoints (p<0.0001). Analysis of skeletal muscle demonstrated significantly greater capillary density in the ischaemic limb of mice treated with iECs, vs. control (179±11 vs 125±10 capillaries/image; p<0.05). In conclusion, iECs have striking in vivo pro-angiogenic effects and appear to bemore potent than iPSC-ECs. These findings support further development of iECs for therapeutic angiogenesis.

Fibroblast growth factor 2 is an essential cardioprotective factor in a closed-chest model of cardiac ischemia-reperfusion injury

Fibroblast growth factor 2 (FGF2) is cardioprotective in in vivo models of myocardial infarction; however, whether FGF2 has a protective role in in vivo ischemia‐reperfusion (IR) injury, a model that more closely mimics acute myocardial infarction in humans, is not known. To assess the cardioprotective efficacy of endogenous FGF2, mice lacking a functional Fgf2 gene (Fgf2−/−) and wild‐type controls were subjected to closed‐chest regional cardiac IR injury (90 min ischemia, 7 days reperfusion). Fgf2−/− mice had significantly increased myocardial infarct size and significantly worsened cardiac function compared to wild‐type controls at both 1 and 7 days post‐IR injury. Pathophysiological analysis showed that at 1 day after IR injury Fgf2−/− mice have worsened cardiac strain patterns and increased myocardial cell death. Furthermore, at 7 days post‐IR injury, Fgf2−/− mice showed a significantly reduced cardiac hypertrophic response, decreased cardiac vessel density, and increased vessel diameter in the peri‐infarct area compared to wild‐type controls. These data reveal both acute cardioprotective and a longer term proangiogenic potential of endogenous FGF2 in a clinically relevant, in vivo, closed‐chest regional cardiac IR injury model that mimics acute myocardial infarction.

Engineered Fibrin Gels for Parallel Stimulation of Mesenchymal Stem Cell Proangiogenic and Osteogenic Potential.

Mesenchymal stem/stromal cells (MSCs) are under examination for use in cell therapies to repair bone defects resulting from trauma or disease. MSCs secrete proangiogenic cues and can be induced to differentiate into bone-forming osteoblasts, yet there is limited evidence that these events can be achieved in parallel. Manipulation of the cell delivery vehicle properties represents a candidate approach for directing MSC function in bone healing. We hypothesized that the biophysical properties of a fibrin gel could simultaneously regulate the proangiogenic and osteogenic potential of entrapped MSCs. Fibrin gels were formed by supplementation with NaCl (1.2, 2.3, and 3.9% w/v) to modulate gel biophysical properties without altering protein concentrations. MSCs entrapped in 1.2% w/v NaCl gels were the most proangiogenic in vitro, yet cells in 3.9% w/v gels exhibited the greatest osteogenic response. Compared to the other groups, MSCs entrapped in 2.3% w/v gels provided the best balance between proangiogenic potential, osteogenic potential, and gel contractility. The contribution of MSCs to bone repair was then examined when deployed in 2.3% w/v NaCl gels and implanted into an irradiated orthotopic bone defect. Compared to acellular gels after 3 weeks of implantation, defects treated with MSC-loaded fibrin gels exhibited significant increases in vessel density, early osteogenesis, superior morphology, and increased cellularity of repair tissue. Defects treated with MSC-loaded gels exhibited increased bone formation after 12 weeks compared to blank gels. These results confirm that fibrin gel properties can be modulated to simultaneously promote both the proangiogenic and osteogenic potential of MSCs, and fibrin gels modified by supplementation with NaCl are promising carriers for MSCs to stimulate bone repair in vivo.

Human Adipose Derived Mesenchymal Stromal Cells Show More Efficient Angiogeneic Promotion on EPCs Than Umbilical Cord and Endometrium

Angiogenesis is a complicated process involving endothelial proliferation, migration, and finally formation of functional vessels, in which perivascular cells play assistant roles. Mesenchymal stem cells (MSCs) from distinct human tissues have been proved to share functional properties and gene-expression profile with perivascular cells and to be proangiogenic. In this study we compared the proangiogenic ability of three types of MSC derived from human adipose stromal cells (ADSCs), umbilical cord (UCMSCs) and endometrium (EMSCs).Three types of MSCs all located around the tubular like structures formed by EPCs in coculture system on Matrigel in vitro (Figure 1A). However, different MSCs showed different potential for stimulating vessel formation of EPCs. After 6 days’ coculture, we found that EPCs seeded on ADSCs monolayer formed more organized capillary-like network (Figure 1B, P<0.01), and the length of vascular, number of junctions and ratio of vascular area was 18.96±3.52, 81.72±14.09 and 23.97%±2.11%, respectively (Figure 1C). EPCs seeded on UCMSCs only formed single tube-like structure (length, 2.90±1.31; junctions, 6.94±3.22; area, 3.05%±1.29%), while EPCs seeded on EMSCs gathered together not to form capillary-like structure (Figure 1B, C).Furthermore, we compared their proangiogenic potential of three types of MSCs. Matrigel contained MSCs and EPCs (ratio 3:2, total 2*106) was injected into nude mouse dorsal subcutaneous. 7 days following the injection, Matrigel was stripped off for the analysis of microvessels. As shown in Figure 2, three types of MSCs all promoted the formation of new microvessels. ADSC had the strongest stimulating effects, and formed more microvessels containing a large number of erythrocytes as shown by the appearance and HE staining (6.43%±2.57%). However, microvessel structures were not observed in the matrigel containing EPCs alone.Moreover, we assessed the therapeutic effects of co-transplantation of EPCs and MSCs in mouse hindlimb ischemic model. Right femoral artery ligation and excision was performed on anesthetized nude mice. After 24h of surgery, EPCs and MSCs were co-transplanted by tail intravenous injection (n=5, total 6.25*105; MSC/EPC ratio, 3:2). Blood flow of the ischemic limb was measured at 0, 7, 14, 21, 28 days after cell transplantation using Laser Doppler Perfusion Imager (Figure 3A). The results showed that the co-transplantation of EPCs with ADSCs significantly improved blood flow of ischemic limb after day 7 of transplantation (0.94±0.10, P<0.01, Figure 3B), and the final limb salvage rate in mice was significantly raised after day 28 of transplantation (Figure 3C, D). Thus, the blood stream reconstitute in early stage is the key factor for the final outcome of ischemic limb.We queried whether the different stimulation effects of MSCs on the vessel formation of EPCs are caused by MSC-secreted proangiogenic cytokines. Firstly, we analyzed the effects of MSC-CM on the proliferation and migration of EPCs in vitro, and found that migration rates/numbers of EPCs was highly increased by ADSC-CM (69.52%±9.66%, P<0.05) compared with UCMSC-CM and EMSC-CM. Next, we analyzed proangiogenic cytokines in MSC-CM using cytokine antibody array. The results showed that ADSCs secreted more proangiogenic cytokines such as VEGF-A, HGF, PDGF-BB and bFGF, and less inhibitor such as thrompospondin-1 in comparision to UCMSCs and EMSCs.In conclusion, MSCs could promote the formation of new microvessles of EPCs, and ADSCs are more efficient than UCMSCs or EMSCs by secreting more proangiogenic cytokines and less inhibitor. Thus ADSCs may be an ideal cellular source for the treatment of ischemia vascular disease. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 17100: Priming with Erythropoietin Enhances Survival and Pro-Angiogenic Properties in Mesenchymal Stem Cells

Introduction: Accumulating evidence from animal studies shows that the administration of mesenchymal stem cells (MSCs) from bone marrow ameliorates tissue damage after ischemic injury. However, the inability to effectively graft culture-expanded stem cells to diseased or injured tissues remains a challenge for cell therapy. Erythropoietin (Epo) is an erythropoiesis-stimulating cytokine and protects erythroid progenitors from cell death. Epo receptor was identified in MSCs, but the action of Epo/Epo receptor signaling is not determined. In the present study we investigated whether Epo enhanced the survival and pro-angiogenic potential in the MCSs in both culture and animal experiments. Methods and Results: Epo receptor was expressed on MSCs isolated from bone marrow in GFP-transgenic rats. In culture study, the Epo treatment (80IU/ml) significantly propagated the MSCs compared with the controls (2-fold increase, p&lt;0.05). Quantitative RT-PCR analysis demonstrated that Epo significantly enhanced the expressions of basic-fibroblast growth factor and stromal cell-derived factor-1 in the cultured MSCs. In vivo, the GFP-MSCs preconditioned with and without Epo (80IU/ml) for 48 hours were locally administered to rat hindlimb ischemia model (n=11 in each group). Priming with Epo significantly increased the MSC engraftment in perivascular area of the injured muscle at day 3 after the implantation more than without Epo (1.5±1.3 vs. 3.0±2.1, p&lt;0.05). The MSCs preconditioned with Epo significantly promoted blood perfusion documented by laser Doppler and capillary growth in histological study compared with the control group at day 14 (p&lt;0.05, respectively). In addition to promoting neovascularization, the MSCs with Epo significantly inhibited macrophage infiltration in perivascular are (p&lt;0.05 vs. the control). Conclusions: Epo induced the proliferative activity and enhanced the production of angiogenic cytokines in the cultured MSCs from bone marrow. In vivo study demonstrated that the short-term priming with Epo promoted the cellular engraftment and neovascularization in the MSC therapy for ischemic limb muscle. MSC implantation combined with Epo may be a novel and feasible strategy in therapeutic angiogeneis.

HDAC-inhibitor (S)-8 disrupts HDAC6-PP1 complex prompting A375 melanoma cell growth arrest and apoptosis.

Histone deacetylase inhibitors (HDACi) are agents capable of inducing growth arrest and apoptosis in different tumour cell types. Previously, we reported a series of novel HDACi obtained by hybridizing SAHA or oxamflatin with 1,4-benzodiazepines. Some of these hybrids proved effective against haematological and solid cancer cells and, above all, compound (S)-8 has emerged for its activities in various biological systems. Here, we describe the effectiveness of (S)-8 against highly metastatic human A375 melanoma cells by using normal PIG1 melanocytes as control. (S)-8 prompted: acetylation of histones H3/H4 and α-tubulin; G0/G1 and G2/M cell cycle arrest by rising p21 and hypophos-phorylated RB levels; apoptosis involving the cleavage of PARP and caspase 9, BAD protein augmentation and cytochrome c release; decrease in cell motility, invasiveness and pro-angiogenic potential as shown by results of wound-healing assay, down-regulation of MMP-2 and VEGF-A/VEGF-R2, besides TIMP-1/TIMP-2 up-regulation; and also intracellular accumulation of melanin and neutral lipids. The pan-caspase inhibitor Z-VAD-fmk, but not the antioxidant N-acetyl-cysteine, contrasted these events. Mechanistically, (S)-8 allows the disruption of cytoplasmic HDAC6-protein phosphatase 1 (PP1) complex in A375 cells thus releasing the active PP1 that dephosphorylates AKT and blocks its downstream pro-survival signalling. This view is consistent with results obtained by: inhibiting PP1 with Calyculin A; using PPP1R2-transfected cells with impaired PP1 activity; monitoring drug-induced HDAC6-PP1 complex re-shuffling; and, abrogating HDAC6 expression with specific siRNA. Altogether, (S)-8 proved very effective against melanoma A375 cells, but not normal melanocytes, and safe to normal mice thus offering attractive clinical prospects for treating this aggressive malignancy.

PPARγ activation but not PPARγ haplodeficiency affects proangiogenic potential of endothelial cells and bone marrow-derived progenitors.

BackgroundPeroxisome proliferator-activated receptor-γ (PPARγ) agonists, which have been used as insulin sensitizers in diabetic patients, may improve functions of endothelial cells (ECs). We investigated the effect of PPARγ on angiogenic activities of murine ECs and bone marrow-derived proangiogenic cells (PACs).MethodsPACs were isolated from bone marrow of 10–12 weeks old, wild type, db/db and PPARγ heterozygous animals. Cells were cultured on fibronectin and gelatin coated dishes in EGM-2MV medium. For in vitro stimulations, rosiglitazone (10 μmol/L) or GW9662 (10 μmol/L) were added to 80% confluent cell cultures for 24 hours. Angiogenic potential of PACs and ECs was tested in vitro and in vivo in wound healing assay and hind limb ischemia model.ResultsECs and PACs isolated from diabetic db/db mice displayed a reduced angiogenic potential in ex vivo and in vitro assays, the effect partially rescued by incubation of cells with rosiglitazone (PPARγ activator). Correction of diabetes by administration of rosiglitazone in vivo did not improve angiogenic potential of isolated PACs or ECs. In a hind limb ischemia model we demonstrated that local injection of conditioned media harvested from wild type PACs improved the blood flow restoration in db/db mice, confirming the importance of paracrine action of the bone marrow-derived cells.Transcriptome analysis showed an upregulation of prooxidative and proinflammatory pathways, and downregulation of several proangiogenic genes in db/db PACs. Interestingly, db/db PACs had also a decreased level of PPARγ and changed expression of PPARγ-regulated genes. Using normoglycemic PPARγ+/− mice we demonstrated that reduced expression of PPARγ does not influence neovascularization either in wound healing or in hind limb ischemia models.ConclusionsIn summary, activation of PPARγ by rosiglitazone improves angiogenic potential of diabetic ECs and PACs, but decreased expression of PPARγ in diabetes does not impair angiogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12933-014-0150-7) contains supplementary material, which is available to authorized users.

Abstract 2365: Analysis of angiogenesis pathway-related gene expression impacted by MDM2 using PCR array

Abstract The Murine Double Minute 2 (MDM2) gene encodes an oncogenic MDM2 protein, which is a negative regulator of the p53 tumor suppressor. MDM2 amplification or over expression is found in many tumors that eventually lead to the inactivation of the cell cycle control and loss of pro-apoptotic functions that are typically exerted by wild type p53. Many of the oncogenic effects of MDM2 are resulting from the inactivation of p53 through direct binding or degradation of p53 following E3 ligase mediated ubiquitination. However, our recent findings have indicated that MDM2 over expression can also promote tumor angiogenesis through p53 independent pathways and thereby enhance the metastatic potential of cancer cells. In this respect increasing the levels of pro-angiogenic factors such as VEGF has been confirmed as one of the mechanisms regulated by MDM2. However, the actual cluster of genes or pathways regulated by MDM2 leading to such increase in the metastatic potentials of cancer cells have not been clearly identified. Therefore, the main purpose of our study was to understand the impact of MDM2 over expression on the levels of angiogenesis related genes that can also increase growth rate and metastatic potential. Hence, for our study we used the Human Angiogenesis PCR Array to compare the gene expression profile of LNCaP (prostate cancer cells) and LNCaP-MST (MDM2 transfected prostate cancer) cells. The MST cells expressed at least 10 fold increase in the levels of MDM2 after transfection. As a result of the elevated levels of MDM2 in LNCaP-MST cells the expression of genes such as THBS1 (125 fold), TNF-α (14 fold), MMP-9 (1.4 fold) were found to be significantly up-regulated compared to the MDM2 non-transfected cells. Cluster analysis further confirmed that these gene expression profiles are positively correlated with the pro-angiogenic potential of the LNCaP-MST cells compared to the non-transfected cells. In addition, the levels of EREG, TIMP1 and CXCL3 were down regulated significantly that favors the suppression of anti-angiogenic mechanisms. Our results offer a conclusive evidence that MDM2 can impact the cancer growth and metastatic potential of cancer cells by shifting the balance towards pro-angiogenic mechanisms (The financial support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida is gratefully acknowledged). Citation Format: Thiagarajan Venkatesan, Corine Stinson, Sivanesan Dhandayuthapani, Appu Rathinavelu. Analysis of angiogenesis pathway-related gene expression impacted by MDM2 using PCR array. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2365. doi:10.1158/1538-7445.AM2014-2365

Activated platelet supernatant can augment the angiogenic potential of human peripheral blood stem cells mobilized from bone marrow by G-CSF

Platelets not only play a role in hemostasis, but they also promote angiogenesis and tissue recovery by releasing various cytokines and making an angiogenic milieu. Here, we examined autologous ‘activated platelet supernatant (APS)’ as a priming agent for stem cells; thereby enhance their pro-angiogenic potential and efficacy of stem cell-based therapy for ischemic diseases. The mobilized peripheral blood stem cells (mobPBSCs) were isolated from healthy volunteers after subcutaneous injection of granulocyte-colony stimulating factor. APS was collected separately from the platelet rich plasma after activation by thrombin. mobPBSCs were primed for 6h before analysis. Compared to naive platelet supernatants, APS had a higher level of various cytokines, such as IL8, IL17, PDGF and VEGF. APS-priming for 6h induced mobPBSCs to express key angiogenic factors, surface markers (i.e. CD34, CD31, and CXCR4) and integrins (integrins α5, β1 and β2). Also mobPBSCs were polarized toward CD14(++)/CD16(+) pro-angiogenic monocytes. The priming effect was reproduced by an in vitro reconstruction of APS. Through this phenotype, APS-priming increased cell–cell adhesion and cell–extracellular matrix adhesion. The culture supernatant of APS-primed mobPBSCs contained high levels of IL8, IL10, IL17 and TNFα, and augmented proliferation and capillary network formation of human umbilical vein endothelial cells. In vivo transplantation of APS-primed mobPBSCs into athymic mice ischemic hindlimbs and Matrigel plugs elicited vessel differentiation and tissue repair. In safety analysis, platelet activity increased after mixing with mobPBSCs regardless of priming, which was normalized by aspirin treatment. Collectively, our data identify that APS-priming can enhance the angiogenic potential of mobPBSCs, which can be used as an adjunctive strategy to improve the efficacy of cell therapy for ischemic diseases.

In vivo angiogenesis screening and mechanism of action of novel tanshinone derivatives produced by one-pot combinatorial modification of natural tanshinone mixture from Salvia miltiorrhiza.

BackgroundNatural products present in low quantity in herb medicines constitute an important source of chemical diversity. However, the isolation of sufficient amounts of these low abundant constituents for structural modification has been a challenge for several decades and subsequently halts research on the utilization of this important source of chemical entities for drug discovery and development. And, pro-angiogenic therapies are being explored as options to treat cardio-cerebral vascular diseases and wound healing recently. The present study investigates the pro-angiogenic potential of tanshinone derivatives produced by one-pot synthesis using zebrafish model.Methodology/Principal FindingsIn order to address the difficulty of chemical modification of low abundant constituents in herb medicines, a novel one-pot combinatorial modification was used to diversify a partially purified tanshinone mixture from Salvia miltiorrhiza. This led to the isolation of ten new imidazole-tanshinones (Compounds 1–10) and one oxazole-tanshinone (Compound 11), the structures of which were characterized by spectroscopic methods in combination with single-crystal X-ray crystallographic analysis. The angiogenesis activities of the new tanshinone derivatives were determined in an experimental model of chemical-induced blood vessels damage in zebrafish. Of all the tested new derivatives, compound 10 exhibited the most potent vascular protective and restorative activity with an EC50 value of 0.026 µM. Moreover, the mechanism underlying the pro-angiogenesis effect of 10 probably involved the VEGF/FGF-Src-MAPK and PI3K-P38 signalling pathways by gene expression analysis and a blocking assay with pathways-specific kinase inhibitors.Conclusions/SignificanceTaken together, our study demonstrated the more distinctive pro-angiogenic properties of 10 than other tanshinones and revealed 10 has potential for development as a pro-angiogenic agent for diseases associated with insufficient angiogenesis. Our results highlighted the great potential of adopting a newly modified one-pot approach to enhance the chemical diversity and biological activities of constituents from natural products regardless of their abundances.

Diabetes Mellitus and Ischemic Diseases

In patients with diabetes mellitus, the ability of ischemic tissue to synchronize the molecular and cellular events leading to restoration of tissue perfusion in response to the atherosclerotic occlusion of a patent artery is markedly impaired. As a consequence, adverse tissue remodeling and the extent of ischemic injury are intensified, leading to increased morbidity and mortality. Growing evidence from preclinical and clinical studies has implicated alterations in hypoxia-inducible factor 1 levels in the abrogation of proangiogenic pathways, including vascular endothelial growth factor A/phosphoinositide 3′ kinase/AKT/endothelial nitric oxide synthase and in the activation of antiangiogenic signals characterized by accumulation of advanced glycation end products, reactive oxygen species overproduction, and endoplasmic reticulum stress. In addition, the diabetic milieu shows a switch toward proinflammatory antiregenerative pathways. Finally, the mobilization, subsequent recruitment, and the proangiogenic potential of the different subsets of angiogenesis-promoting bone marrow–derived cells are markedly impaired in the diabetic environment. In this review, we will give an overview of the current understanding on the signaling molecules contributing to the diabetes mellitus–induced impairment of postischemic revascularization mainly in the setting of myocardial infarction or critical limb ischemia.

Engineering a vascular endothelial growth factor 165-binding heparan sulfate for vascular therapy

The therapeutic use of VEGF165 to stimulate blood vessel formation for the treatment of peripheral arterial disease or cardiovascular-related disease has met with limited success. Here we describe an affinity-isolated heparan sulfate glycotherapeutic (HS7+ve) that binds to, and enhances the bioactivity of, VEGF165. Application of HS7+ve complexed with VEGF165 results in enhanced VEGF165–VEGFR2 interaction, prolonged downstream pErk1/2 signalling, and increased cell proliferation and tube formation in HUVECs, compared with VEGF165 alone. The pro-angiogenic potential of HS7+ve was further assessed in vivo using the chick embryo chorioallantoic membrane (CAM) assay. Exogenous dosing with HS7+ve alone significantly enhanced the formation of new blood vessels with potencies comparable to VEGF165. These results demonstrate the potential for vascular therapy of glycotherapeutic agents targeted at augmenting the bioactivity of VEGF165.

Human mesenchymal stem cell spheroids in fibrin hydrogels exhibit improved cell survival and potential for bone healing.

Mesenchymal stem cells (MSCs) have great therapeutic potential for the repair of nonhealing bone defects, because of their proliferative capacity, multilineage potential, trophic factor secretion and lack of immunogenicity. However, a major challenge to the translation of cell-based therapies into clinical practice is ensuring their survival and function upon implantation into the defect site. We hypothesize that forming MSCs into more physiologic three-dimensional spheroids, rather than employing dissociated cells from two-dimensional monolayer culture, will enhance their survival when exposed to a harsh microenvironment but maintain their osteogenic potential. MSC spheroids were formed by using the hanging drop method with increasing cell numbers. Compared with larger spheroids, the smallest spheroids, which contained 15,000 cells, exhibited increased metabolic activity, reduced apoptosis and the most uniform distribution of proliferating cells. Spheroids were then entrapped in fibrin gels and cultured in serum-free medium and 1% oxygen. Compared with identical numbers of dissociated MSCs in fibrin gels, spheroids exhibited significantly reduced apoptosis and secreted up to 100-fold more vascular endothelial growth factor. Moreover, fibrin gels containing spheroids and those containing an equivalent number of dissociated cells exhibited similar expression levels of early and late markers of osteogenic differentiation. Thus, MSC spheroids exhibit greater resistance to apoptosis and enhanced proangiogenic potential while maintaining similar osteogenic potential to dissociated MSCs entrapped in a clinically relevant biomaterial, supporting the use of MSC spheroids in cell-based approaches to bone repair.

Candesartan Induces a Prolonged Proangiogenic Effect and Augments Endothelium-Mediated Neuroprotection after Oxygen and Glucose Deprivation: Role of Vascular Endothelial Growth Factors A and B

Angiogenesis is a key component of recovery after stroke. Angiotensin II receptor blocker (ARB) treatment improves neurobehavioral outcome and is associated with enhanced angiogenesis after stroke. The purpose of this study is to investigate the temporal pattern of the ARB proangiogenic effect in the ischemic brain and its association with vascular endothelial growth factors VEGF-A and VEGF-B. Wistar rats were exposed to 90-minute middle cerebral artery occlusion and treated with candesartan (1 mg/kg) at reperfusion. The proangiogenic potential of the cerebrospinal fluid was determined at 8, 24, 48, and 72 hours using an in vitro Matrigel tube formation assay. In addition, the expression of VEGF-A and VEGF-B was measured in brain homogenates using Western blotting at the same time points. A single candesartan dose induced a prolonged proangiogenic effect and a prolonged upregulation of VEGF-A and VEGF-B in vivo. In the ischemic hemisphere, candesartan treatment was associated with stabilization of hypoxia-inducible factor-1α and preservation of angiopoietin-1. The effect of ARB treatment on endothelial cells was studied in vitro. Our results identified brain endothelial cells as one target for the action of ARBs and a source of the upregulated VEGF-A and VEGF-B, which exerted an autocrine angiogenic response, in addition to a paracrine neuroprotective effect. Taken together, this study highlights the potential usefulness of augmenting the endogenous restorative capacity of the brain through the administration of ARBs.

Dynamic induction of pro-angiogenic milieu after transplantation of marrow-derived mesenchymal stem cells in experimental myocardial infarction

BackgroundCell-based pro-angiogenic therapy by bone marrow mesenchymal stem cells (MSCs) has been touted as a means to reducing the adverse effects of cardiac remodeling after myocardial infarction (MI). Milieu-dependent regulation of pro-angiogenic potential of MSCs after infarction remains to be elucidated. In this study, the effects of marrow-derived MSCs on the kinetics of angiogenesis signaling factors were investigated in a rabbit model of MI. MethodsMI was induced in rabbits, and the animals were randomized into two groups (cell transplantation and control, each group with 21 animals). 1×106 autologous marrow-derived MSCs were injected into the myocardium of the border zone after transfection with a green fluorescent protein (GFP) lentiviral reporter vector. Control animals received PBS vehicle only. Effect of the transplanted cells on the hearts was evaluated over time by pathological, immunofluorescence, western blotting, immuno-electron microscopy, and echocardiographic analyses. ResultsTransplanted GFP-positive MSCs were enriched with time in the peri-infarct border zone with differentiation potential into three major cell types of the heart, including cardiomyocytes, endothelial cells, and smooth muscle cells, and there was significant augmentation of microvascular density. The transplanted cells could change the milieu of the injured myocardium to increase the expression levels of VEGF as well as the ratio of Ang-2 to Ang-1, and to reduce the ratio of phosphorylated Tie2 to Tie2. ConclusionAn angiogenesis-promoting milieu was induced after the transplantation of marrow MSCs in the injured myocardium. Compared with the resident cells, the transplanted cells had a greater rate of cellular kinetics in the infarcted myocardium.

Heme Oxygenase-1 Is Required for Angiogenic Function of Bone Marrow-Derived Progenitor Cells: Role in Therapeutic Revascularization

Heme oxygenase-1 (HO-1) is a cytoprotective enzyme that can be down-regulated in diabetes. Its importance for mature endothelium has been described, but its role in proangiogenic progenitors is not well known. We investigated the effect of HO-1 on the angiogenic potential of bone marrow-derived cells (BMDCs) and on blood flow recovery in ischemic muscle of diabetic mice. Lack of HO-1 decreased the number of endothelial progenitor cells (Lin(-)CD45(-)cKit(-)Sca-1(+)VEGFR-2(+)) in murine bone marrow, and inhibited the angiogenic potential of cultured BMDCs, affecting their survival under oxidative stress, proliferation, migration, formation of capillaries, and paracrine proangiogenic potential. Transcriptome analysis of HO-1(-/-) BMDCs revealed the attenuated up-regulation of proangiogenic genes in response to hypoxia. Heterozygous HO-1(+/-) diabetic mice subjected to hind limb ischemia exhibited reduced local expression of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), stromal cell-derived factor 1 (SDF-1), VEGFR-1, VEGFR-2, and CXCR-4. This was accompanied by impaired revascularization of ischemic muscle, despite a strong mobilization of bone marrow-derived proangiogenic progenitors (Sca-1(+)CXCR-4(+)) into peripheral blood. Blood flow recovery could be rescued by local injections of conditioned media harvested from BMDCs, but not by an injection of cultured BMDCs. This is the first report showing that HO-1 haploinsufficiency impairs tissue revascularization in diabetes and that proangiogenic in situ response, not progenitor cell mobilization, is important for blood flow recovery. HO-1 is necessary for a proper proangiogenic function of BMDCs. A low level of HO-1 in hyperglycemic mice decreases restoration of perfusion in ischemic muscle, which can be rescued by a local injection of conditioned media from cultured BMDCs.

Novel and Emerging Therapies for Pulmonary Hypertension

The development of therapeutic concepts in pulmonary hypertension (PH) is intimately linked with the unraveling of pathogenetic sequelae. This perspective highlights advances in our understanding of the regulation of vasomotion and vascular remodeling that have led to "reverse-remodeling" and regenerative strategies as novel treatment concepts. Progress has been made in understanding redox-dependent signaling; inflammatory sequelae; and transcription factor, ion channel, and metabolic abnormalities, as well as growth factor-dependent hyperproliferation that underlies PH. We are, however, far from understanding the molecular pathways that differentially drive the various vascular phenotypes (intimal thickening, media hypertrophy, adventitial thickening, plexiform lesions, vascular pruning) in this disease. Antiproliferative strategies, transcription factor-based therapies, inflammation/immune cell-focused approaches, and epigenetic modulation-based therapies are all novel treatment concepts for PH. The proangiogenic potential of genetically engineered mesenchymal stem cells and endothelial progenitor cells has been explored as a regenerative strategy. The progress that has been made in identifying important cellular and molecular mechanisms and applying this knowledge to novel therapies is largely restricted to group 1 PH. However, understanding the molecular sequelae underlying PH in groups 2 through 5 PH is also urgently needed.

Enhanced trophic factor secretion by mesenchymal stem/stromal cells with Glycine-Histidine-Lysine (GHK)-modified alginate hydrogels

Recombinant proteins and cytokines are under broad preclinical and clinical investigation to promote angiogenesis, but their success is limited by ineffective delivery, lack of long-term stability and excessive cost. Mesenchymal stem/stromal cells (MSC) secrete bioactive trophic factors, and thus, may provide an effective alternative to address these challenges. Glycine-Histidine-Lysine (GHK) is a peptide fragment of osteonectin, a matricellular protein with reported proangiogenic potential. We examined the capacity of GHK to up-regulate secretion of proangiogenic factors from human MSC in culture and when covalently coupled to alginate hydrogels. GHK had no apparent cytotoxic effects on MSC in culture over a wide range of concentrations. We detected a dose-dependent increase in vascular endothelial growth factor (VEGF) concentration in media conditioned by GHK-treated MSC, which increased endothelial cell proliferation, migration and tubule formation. We covalently coupled GHK to alginate using carbodiimide chemistry, and human MSC were entrapped in alginate hydrogels to assess VEGF secretion. Similar to monolayer culture, MSC responded to GHK-modified gels by secreting increased concentrations of VEGF and basic fibroblast growth factor compared to unmodified gels. The pre-treatment of MSC with antibodies to α6 and β1 integrins prior to entrapment in GHK-modified gels abrogated VEGF secretion, suggesting that the proangiogenic response of MSC was integrin-mediated. These data demonstrate that the proangiogenic potential of MSC can be significantly increased by the presentation of GHK with a biodegradable carrier, therefore increasing their clinical potential when used for tissue repair.

Angiotensin II-Derived Reactive Oxygen Species Promote Angiogenesis in Human Late Endothelial Progenitor Cells Through Heme Oxygenase-1 via ERK1/2 and AKT/PI3K Pathways

Angiotensin II (Ang II), the main component of renin-angiotensin system, could mediate pathogenic angiogenesis in cardiovascular disorders. Late endothelial progenitor cells (EPCs) possess potent self-renewal and angiogenic potency superior to early EPCs, but few study focused on the cross-talk between Ang II and late EPCs. We observed that Ang II could increase reactive oxygen species (ROS) and promote capillary formation in late EPCs. Ang II-derived ROS could also upregulate heme oxygenase-1 (HO-1) expression, and treating late EPCs with HO-1 small interfering RNA or heme oxygenase inhibitor (HO inhibitor) could inhibit Ang II-induced tube formation and increase ROS level and apoptosis rate. In addition, PD98059 and LY294002 pretreatment attenuated Ang II-induced HO-1 expression. Accordingly, Ang II-derived ROS could promote angiogenesis in late EPCs by inducing HO-1 expression via ERK1/2 and AKT/PI3K pathways, and we believe HO-1 might be a promising intervention target in EPCs due to its potent proangiogenic, antioxidant, and antiapoptosis potentials.