PlGF repairs myocardial ischemia through mechanisms of angiogenesis, cardioprotection and recruitment of myo-angiogenic competent marrow progenitors

Rationale

Despite preclinical success in regenerating and revascularizing the infarcted heart using angiogenic growth factors or bone marrow (BM) cells, recent clinical trials have revealed less benefit from these therapies than expected.

Objective

We explored the therapeutic potential of myocardial gene therapy of placental growth factor (PlGF), a VEGF-related angiogenic growth factor, with progenitor-mobilizing activity.

Methods and Results

Myocardial PlGF gene therapy improves cardiac performance after myocardial infarction, by inducing cardiac repair and reparative myoangiogenesis, via upregulation of paracrine anti-apoptotic and angiogenic factors. In addition, PlGF therapy stimulated Sca-1⁺/Lin⁻ (SL) BM progenitor proliferation, enhanced their mobilization into peripheral blood, and promoted their recruitment into the peri-infarct borders. Moreover, PlGF enhanced endothelial progenitor colony formation of BM-derived SL cells, and induced a phenotypic switch of BM-SL cells, recruited in the infarct, to the endothelial, smooth muscle and cardiomyocyte lineage.

Conclusions

Such pleiotropic effects of PlGF on cardiac repair and regeneration offer novel opportunities in the treatment of ischemic heart disease.     

Pleiotropic effects of statins in distal human pulmonary artery smooth muscle cells

Background

Recent clinical data suggest statins have transient but significant effects in patients with pulmonary arterial hypertension. In this study we explored the molecular effects of statins on distal human pulmonary artery smooth muscle cells (PASMCs) and their relevance to proliferation and apoptosis in pulmonary arterial hypertension.

Methods

Primary distal human PASMCs from patients and controls were treated with lipophilic (simvastatin, atorvastatin, mevastatin and fluvastatin), lipophobic (pravastatin) and nitric-oxide releasing statins and studied in terms of their DNA synthesis, proliferation, apoptosis, matrix metalloproteinase-9 and endothelin-1 release.

Results

Treatment of human PASMCs with selected statins inhibited DNA synthesis, proliferation and matrix metalloproteinase-9 production in a concentration-dependent manner. Statins differed in their effectiveness, the rank order of anti-mitogenic potency being simvastatin > atorvastatin > > pravastatin. Nevertheless, a novel nitric oxide-releasing derivative of pravastatin (NCX 6550) was effective. Lipophilic statins, such as simvastatin, also enhanced the anti-proliferative effects of iloprost and sildenafil, promoted apoptosis and inhibited the release of the mitogen and survival factor endothelin-1. These effects were reversed by mevalonate and the isoprenoid intermediate geranylgeranylpyrophosphate and were mimicked by inhibitors of the Rho and Rho-kinase.

Conclusions

Lipophilic statins exert direct effects on distal human PASMCs and are likely to involve inhibition of Rho GTPase signalling. These findings compliment some of the recently documented effects in patients with pulmonary arterial hypertension.     

The 3-hydroxy-3-methylglutaryl co-enzyme A reductase inhibitor pravastatin enhances neurite outgrowth in hippocampal neurons

Epidemiological studies demonstrate a relationship between statin [3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor] usage and reduced risk of developing Alzheimer's disease. To determine whether statins affect neuronal development, we treated cultured rat hippocampal neurons with pravastatin. After 4-48 h of treatment, pravastatin significantly increased the number of neurites produced by each cell and caused a corresponding increase in levels of the membrane phospholipid phosphatidylcholine. Pravastatin treatment also significantly increased neurite length and branching but did not affect cellular cholesterol levels. Co-incubation with mevalonate, but not cholesterol, abolished the stimulatory effect of pravastatin on neurite outgrowth. Treatment of neurons with isoprenoids also abolished the effect of pravastatin on neurite growth, suggesting that pravastatin may stimulate neuritogenesis by preventing isoprenylation of signaling molecules such as the Rho family of small GTPases. A specific inhibitor of geranylgeranylation, but not farnesylation, mimicked the stimulatory effect of pravastatin on neuritogenesis. Pravastatin treatment significantly decreased levels of membrane-associated RhoA. These data suggest that pravastatin treatment increases neurite outgrowth and may do so via inhibiting the activity of geranylgeranylated proteins such as RhoA.     

Matrix Conditions and KLF2-Dependent Induction of Heme Oxygenase-1 Modulate Inhibition of HCV Replication by Fluvastatin

Background & Aims

HMG-CoA-reductase-inhibitors (statins) have been shown to interfere with HCV replication in vitro. We investigated the mechanism, requirements and contribution of heme oxygenase-1(HO-1)-induction by statins to interference with HCV replication.

Methods

HO-1-induction by fluva-, simva-, rosuva-, atorva- or pravastatin was correlated to HCV replication, using non-infectious replicon systems as well as the infectious cell culture system. The mechanism of HO-1-induction by statins as well as its relevance for interference with HCV replication was investigated using transient or permanent knockdown cell lines. Polyacrylamide(PAA) gels of different density degrees or the Rho-kinase-inhibitor Hydroxyfasudil were used in order to mimic matrix conditions corresponding to normal versus fibrotic liver tissue.

Results

All statins used, except pravastatin, decreased HCV replication and induced HO-1 expression, as well as interferon response in vitro. HO-1-induction was mediated by reduction of Bach1 expression and induction of the Nuclear factor (erythroid-derived 2)-like 2 (NRF2) cofactor Krueppel-like factor 2 (KLF2). Knockdown of KLF2 or HO-1 abrogated effects of statins on HCV replication. HO-1-induction and anti-viral effects of statins were more pronounced under cell culture conditions mimicking advanced stages of liver disease.

Conclusions

Statin-mediated effects on HCV replication seem to require HO-1-induction, which is more pronounced in a microenvironment resembling fibrotic liver tissue. This implicates that certain statins might be especially useful to support HCV therapy of patients at advanced stages of liver disease.     

Combinatorial G-CSF/AMD3100 Treatment in Cardiac Repair after Myocardial Infarction

Aims

Several studies suggest that circulating bone marrow derived stem cells promote the regeneration of ischemic tissues. For hematopoietic stem cell transplantation combinatorial granulocyte-colony stimulating factor (G-CSF)/Plerixafor (AMD3100) administration was shown to enhance mobilization of bone marrow derived stem cells compared to G-CSF monotherapy. Here we tested the hypothesis whether combinatorial G-CSF/AMD3100 therapy has beneficial effects in cardiac recovery in a mouse model of myocardial infarction.

Methods

We analyzed the effect of single G-CSF (250 µg/kg/day) and combinatorial G-CSF/AMD3100 (100 µg/kg/day) treatment on cardiac morphology, vascularization, and hemodynamics 28 days after permanent ligation of the left anterior descending artery (LAD). G-CSF treatment started directly after induction of myocardial infarction (MI) for 3 consecutive days followed by a single AMD3100 application on day three after MI in the G-CSF/AMD3100 group. Cell mobilization was assessed by flow cytometry of blood samples drawn from tail vein on day 0, 7, and 14.

Results

Peripheral blood analysis 7 days after MI showed enhanced mobilization of white blood cells (WBC) and endothelial progenitor cells (EPC) upon G-CSF and combinatorial G-CSF/AMD3100 treatment. However, single or combinatorial treatment showed no improvement in survival, left ventricular function, and infarction size compared to the saline treated control group 28 days after MI. Furthermore, no differences in histology and vascularization of infarcted hearts could be observed.

Conclusion

Although the implemented treatment regimen caused no adverse effects, our data show that combinatorial G-CSF/AMD therapy does not promote myocardial regeneration after permanent LAD occlusion.     

Augmentation of Neovascularizaiton in Hindlimb Ischemia by Combined Transplantation of Human Embryonic Stem Cells-Derived Endothelial and Mural Cells

Background

We demonstrated that mouse embryonic stem (ES) cells-derived vascular endothelial growth factor receptor-2 (VEGF-R2) positive cells could differentiate into both endothelial cells (EC) and mural cells (MC), and termed them as vascular progenitor cells (VPC). Recently, we have established a method to expand monkey and human ES cells-derived VPC with the proper differentiation stage in a large quantity. Here we investigated the therapeutic potential of human VPC-derived EC and MC for vascular regeneration.

Methods and Results

After the expansion of human VPC-derived vascular cells, we transplanted these cells to nude mice with hindlimb ischemia. The blood flow recovery and capillary density in ischemic hindlimbs were significantly improved in human VPC-derived EC-transplanted mice, compared to human peripheral and umbilical cord blood-derived endothelial progenitor cells (pEPC and uEPC) transplanted mice. The combined transplantation of human VPC-derived EC and MC synergistically improved blood flow of ischemic hindlimbs remarkably, compared to the single cell transplantations. Transplanted VPC-derived vascular cells were effectively incorporated into host circulating vessels as EC and MC to maintain long-term vascular integrity.

Conclusions

Our findings suggest that the combined transplantation of human ES cells-derived EC and MC can be used as a new promising strategy for therapeutic vascular regeneration in patients with tissue ischemia.     

Studies on the antibacterial effects of statins--in vitro and in vivo

Background

Statin treatment has been associated with a beneficial outcome on respiratory tract infections. In addition, previous in vitro and in vivo experiments have indicated favorable effects of statins in bacterial infections.

Aim

The aim of the present study was to elucidate possible antibacterial effects of statins against primary pathogens of the respiratory tract.

Methods

MIC-values for simvastatin, fluvastatin and pravastatin against S. pneumoniae, M. catarrhalis and H. influenzae were determined by traditional antibacterial assays. A BioScreen instrument was used to monitor effects of statins on bacterial growth and to assess possible synergistic effects with penicillin. Bacterial growth in whole blood and serum from healthy volunteers before and after a single dose of simvastatin, fluvastatin and penicillin (positive control) was determined using a blood culture system (BactAlert).

Findings

The MIC-value for simvastatin against S pneumoniae and M catarrhalis was 15 µg/mL (36 mmol/L). Fluvastatin and Pravastatin showed no antibacterial effect in concentrations up to 100 µg/mL (230 µmol/L). Statins did not affect growth or viability of H influenzae. Single doses of statins given to healthy volunteers did not affect growth of pneumococci, whereas penicillin efficiently killed all bacteria.

Conclusions

Simvastatin at high concentrations 15 µg/mL (36 µmol/L) rapidly kills S pneumoniae and M catarrhalis. However, these concentrations by far exceed the concentrations detected in human blood during simvastatin therapy (1–15 nmol/L) and single doses of statins given to healthy volunteers did not improve antibacterial effects of whole blood. Thus, a direct bactericidal effect of statins in vivo is probably not the mechanism behind the observed beneficial effect of statins against various infections.     

The potential of statin and stromal cell-derived factor-1 to promote angiogenesis

Angiogenesis requires the mobilization of progenitor cells from the bone marrow (BM) and homing of progenitor cells to ischemic tissue. The cholesterol lowering drug Statins can stimulate angiogenesis via mobilization of BM derived endothelial progenitor cells (EPCs), promoting EPC migration, and inhibiting EPC apoptosis. The chemokine stromal cell-derived factor-1 (SDF-1) augments EPC chemotaxis, facilitates EPC incorporation into the neovasculature. The combined use of a statin to mobilize EPCs and local over-expression of SDF-1 to augment EPC homing to ischemic muscle resulted in superior angiogenesis versus use of either agent alone. Their effects are through augmenting EPC mobilization, incorporation, proliferation, migration, and tube formation while inhibiting EPC apoptosis. Statin and SDF-1 therefore display synergism in promoting neovascularization by improving reperfusion of ischemic muscle, increasing progenitor cell presentation and capillary density in ischemic muscle, and diminishing apoptosis. These results suggest that the combination of statin and SDF-1 may be a new therapeutic strategy in the treatment of limb ischemia.     

Ultrastructural Features of Ischemic Tissue following Application of a Bio-Membrane Based Progenitor Cardiomyocyte Patch for Myocardial Infarction Repair

Background and Objective

Implantation of cell-sheets into damaged regions of the heart after myocardial infarction (MI) has been shown to improve heart function. However, the tissue morphology following application of induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) has not been studied in detail at the level afforded by electron microscopy. We hypothesized that increasing the number of CM derived from iPSC would increase the effectiveness of cell-sheets used to treat ischemic cardiomyopathy. We report here on the ultrastructural features after application of a bio-membrane ‘cell patch’.

Methods

iPSC-derived progenitor cells were transduced using lentivirus vectors with or without NCX1 promoter. iPSC-CM sheets were transplanted over the transmural MI region in a mouse model of regional ischemic cardiomyopathy. Mice were divided into four groups, 1) Sham; 2) MI; 3) MI + iPSC without NCX1 treated cells (MI + iPSC^Null) and 4) MI + iPSC receiving NCX1 promoter treated cells (MI + iPSC^NCX1). Echocardiography was performed 4 weeks after cell patch application, followed by histological and transmission electron microscopy (TEM) analysis.

Results

Large numbers of transplanted CM were observed with significant improvements in left ventricular performance and remodeling in group 4 as compared with group 3. No teratoma formation was detected in any of the treatment groups.

Conclusion

Manipulation of iPSC yields large numbers of iPSC-CM and favorable morphological and ultrastructural tissue changes. These changes have the potential to enhance current methods used for restoration of cardiac function after MI.     

Validation of the Cardiosphere Method to Culture Cardiac Progenitor Cells from Myocardial Tissue

Background

At least four laboratories have shown that endogenous cardiac progenitor cells (CPCs) can be grown directly from adult heart tissue in primary culture, as cardiospheres or their progeny (cardiosphere-derived cells, CDCs). Indeed, CDCs are already being tested in a clinical trial for cardiac regeneration. Nevertheless, the validity of the cardiosphere strategy to generate CPCs has been called into question by reports based on variant methods. In those reports, cardiospheres are argued to be cardiomyogenic only because of retained cardiomyocytes, and stem cell activity has been proposed to reflect hematological contamination. We use a variety of approaches (including genetic lineage tracing) to show that neither artifact is applicable to cardiospheres and CDCs grown using established methods, and we further document the stem cell characteristics (namely, clonogenicity and multilineage potential) of CDCs.

Methodology/Principal Findings

CPCs were expanded from human endomyocardial biopsies (n = 160), adult bi-transgenic MerCreMer-Z/EG mice (n = 6), adult C57BL/6 mice (n = 18), adult GFP⁺ C57BL/6 transgenic mice (n = 3), Yucatan mini pigs (n = 67), adult SCID beige mice (n = 8), and adult Wistar-Kyoto rats (n = 80). Cellular yield was enhanced by collagenase digestion and process standardization; yield was reduced in altered media and in specific animal strains. Heparinization/retrograde organ perfusion did not alter the ability to generate outgrowth from myocardial sample. The initial outgrowth from myocardial samples was enriched for sub-populations of CPCs (c-Kit⁺), endothelial cells (CD31⁺, CD34⁺), and mesenchymal cells (CD90⁺). Lineage tracing using MerCreMer-Z/EG transgenic mice revealed that the presence of cardiomyocytes in the cellular outgrowth is not required for the generation of CPCs. Rat CDCs are shown to be clonogenic, and cloned CDCs exhibit spontaneous multineage potential.

Conclusions/Significance

This study demonstrates that direct culture and expansion of CPCs from myocardial tissue is simple, straightforward, and reproducible when appropriate techniques are used.     

Molecular Strategy to Reduce In Vivo Collagen Barrier Promotes Entry of NCX1 Positive Inducible Pluripotent Stem Cells (iPSCNCX1+) into Ischemic (or Injured) Myocardium

Objective

The purpose of this study was to assess the effect of collagen composition on engraftment of progenitor cells within infarcted myocardium.

Background

We previously reported that intramyocardial penetration of stem/progenitor cells in epicardial patches was enhanced when collagen was reduced in hearts overexpressing adenylyl cyclase-6 (AC6). In this study we hypothesized an alternative strategy wherein overexpression of microRNA-29b (miR-29b), inhibiting mRNAs that encode cardiac fibroblast proteins involved in fibrosis, would similarly facilitate progenitor cell migration into infarcted rat myocardium.

Methods

In vitro: A tri-cell patch (Tri-P) consisting of cardiac sodium-calcium exchanger-1 (NCX1) positive iPSC (iPSC^NCX1+), endothelial cells (EC), and mouse embryonic fibroblasts (MEF) was created, co-cultured, and seeded on isolated peritoneum. The expression of fibrosis-related genes was analyzed in cardiac fibroblasts (CFb) by qPCR and Western blot. In vivo: Nude rat hearts were administered mimic miRNA-29b (miR-29b), miRNA-29b inhibitor (Anti-29b), or negative mimic (Ctrl) before creation of an ischemically induced regional myocardial infarction (MI). The Tri-P was placed over the infarcted region 7 days later. Angiomyogenesis was analyzed by micro-CT imaging and immunofluorescent staining. Echocardiography was performed weekly.

Results

The number of green fluorescent protein positive (GFP⁺) cells, capillary density, and heart function were significantly increased in hearts overexpressing miR-29b as compared with Ctrl and Anti-29b groups. Conversely, down-regulation of miR-29b with anti-29b in vitro and in vivo induced interstitial fibrosis and cardiac remodeling.

Conclusion

Overexpression of miR-29b significantly reduced scar formation after MI and facilitated iPSC^NCX1+ penetration from the cell patch into the infarcted area, resulting in restoration of heart function after MI.     

Lung-Homing of Endothelial Progenitor Cells and Airway Vascularization Is Only Partially Dependant on Eosinophils in a House Dust Mite-Exposed Mouse Model of Allergic Asthma

Background

Asthmatic responses involve a systemic component where activation of the bone marrow leads to mobilization and lung-homing of progenitor cells. This traffic may be driven by stromal cell derived factor-1 (SDF-1), a potent progenitor chemoattractant. We have previously shown that airway angiogenesis, an early remodeling event, can be inhibited by preventing the migration of endothelial progenitor cells (EPC) to the lungs. Given intranasally, AMD3100, a CXCR4 antagonist that inhibits SDF-1 mediated effects, attenuated allergen-induced lung-homing of EPC, vascularization of pulmonary tissue, airway eosinophilia and development of airway hyperresponsiveness. Since SDF-1 is also an eosinophil chemoattractant, we investigated, using a transgenic eosinophil deficient mouse strain (PHIL) whether EPC lung accumulation and lung vascularization in allergic airway responses is dependent on eosinophilic inflammation.

Methods

Wild-type (WT) BALB/c and eosinophil deficient (PHIL) mice were sensitized to house dust mite (HDM) using a chronic exposure protocol and treated with AMD3100 to modulate SDF-1 stimulated progenitor traffic. Following HDM challenge, lung-extracted EPCs were enumerated along with airway inflammation, microvessel density (MVD) and airway methacholine responsiveness (AHR).

Results

Following Ag sensitization, both WT and PHIL mice exhibited HDM-induced increase in airway inflammation, EPC lung-accumulation, lung angiogenesis and AHR. Treatment with AMD3100 significantly attenuated outcome measures in both groups of mice. Significantly lower levels of EPC and a trend for lower vascularization were detected in PHIL versus WT mice.

Conclusions

This study shows that while allergen-induced lung-homing of endothelial progenitor cells, increased tissue vascularization and development lung dysfunction can occur in the absence of eosinophils, the presence of these cells worsens the pathology of the allergic response.     

Cardiosphere-derived cells improve function in the infarcted rat heart for at least 16 weeks--an MRI study

Aims

Endogenous cardiac progenitor cells, expanded from explants via cardiosphere formation, present a promising cell source to prevent heart failure following myocardial infarction. Here we used cine-magnetic resonance imaging (MRI) to track administered cardiosphere-derived cells (CDCs) and to measure changes in cardiac function over four months in the infarcted rat heart.

Methods and Results

CDCs, cultured from neonatal rat heart, comprised a heterogeneous population including cells expressing the mesenchymal markers CD90 and CD105, the stem cell marker c-kit and the pluripotency markers Sox2, Oct3/4 and Klf-4. CDCs (2×10⁶) expressing green fluorescent protein (GFP+) were labelled with fluorescent micron-sized particles of iron oxide (MPIO). Labelled cells were administered to the infarcted rat hearts (n = 7) by intramyocardial injection immediately following reperfusion, then by systemic infusion (4×10⁶) 2 days later. A control group (n = 7) was administered cell medium. MR hypointensities caused by the MPIOs were detected at all times and GFP+ cells containing MPIO particles were identified in tissue slices at 16 weeks. At two days after infarction, cardiac function was similar between groups. By 6 weeks, ejection fractions in control hearts had significantly decreased (47±2%), but this was not evident in CDC-treated hearts (56±3%). The significantly higher ejection fractions in the CDC-treated group were maintained for a further 10 weeks. In addition, CDC-treated rat hearts had significantly increased capillary density in the peri-infarct region and lower infarct sizes. MPIO-labelled cells also expressed cardiac troponin I, von Willebrand factor and smooth muscle actin, suggesting their differentiation along the cardiomyocyte lineage and the formation of new blood vessels.

Conclusions

CDCs were retained in the infarcted rat heart for 16 weeks and improved cardiac function.     

Zoledronate inhibits ischemia-induced neovascularization by impairing the mobilization and function of endothelial progenitor cells

Background

Bisphosphonates are a class of pharmacologic compounds that are commonly used to treat postmenopausal osteoporosis and malignant osteolytic processes. Studies have shown that bone marrow-derived endothelial progenitor cells (EPCs) play a significant role in postnatal neovascularization. Whether the nitrogen-containing bisphosphonate zoledronate inhibits ischemia-induced neovascularization by modulating EPC functions remains unclear.

Methodology/Principal Findings

Unilateral hindlimb ischemia was surgically induced in wild-type mice after 2 weeks of treatment with vehicle or zoledronate (low-dose: 30 μg/kg; high-dose: 100 μg/kg). Doppler perfusion imaging demonstrated that the ischemic limb/normal side blood perfusion ratio was significantly lower in wild-type mice treated with low-dose zoledronate and in mice treated with high-dose zoledronate than in controls 4 weeks after ischemic surgery (control vs. low-dose vs. high-dose: 87±7% vs. *61±18% vs. **49±17%, *p<0.01, **p<0.005 compared to control). Capillary densities were also significantly lower in mice treated with low-dose zoledronate and in mice treated with high-dose zoledronate than in control mice. Flow cytometry analysis showed impaired mobilization of EPC-like cells (Sca-1⁺/Flk-1⁺) after surgical induction of ischemia in mice treated with zoledronate but normal levels of mobilization in mice treated with vehicle. In addition, ischemic tissue from mice that received zoledronate treatment exhibited significantly lower levels of the active form of MMP-9, lower levels of VEGF, and lower levels of phosphorylated eNOS and phosphorylated Akt than ischemic tissue from mice that received vehicle. Results of the in vitro studies showed that incubation with zoledronate inhibited the viability, migration, and tube-forming capacities of EPC.

Conclusions/Significance

Zoledronate inhibited ischemia-induced neovascularization by impairing EPC mobilization and angiogenic functions. These findings suggest that administration of zoledronate should be withheld in patients with ischemic events such as acute limb ischemia.     

Sca-1+ cardiosphere-derived cells are enriched for Isl1-expressing cardiac precursors and improve cardiac function after myocardial injury

Background

Endogenous cardiac progenitor cells are a promising option for cell-therapy for myocardial infarction (MI). However, obtaining adequate numbers of cardiac progenitors after MI remains a challenge. Cardiospheres (CSs) have been proposed to have cardiac regenerative properties; however, their cellular composition and how they may be influenced by the tissue milieu remains unclear.

Methodology/Principal Finding

Using “middle aged” mice as CSs donors, we found that acute MI induced a dramatic increase in the number of CSs in a mouse model of MI, and this increase was attenuated back to baseline over time. We also observed that CSs from post-MI hearts engrafted in ischemic myocardium induced angiogenesis and restored cardiac function. To determine the role of Sca-1⁺CD45^- cells within CSs, we cloned these from single cell isolates. Expression of Islet-1 (Isl1) in Sca-1⁺CD45^- cells from CSs was 3-fold higher than in whole CSs. Cloned Sca-1⁺CD45^- cells had the ability to differentiate into cardiomyocytes, endothelial cells and smooth muscle cells in vitro. We also observed that cloned cells engrafted in ischemic myocardium induced angiogenesis, differentiated into endothelial and smooth muscle cells and improved cardiac function in post-MI hearts.

Conclusions/Significance

These studies demonstrate that cloned Sca-1⁺CD45^- cells derived from CSs from infarcted “middle aged” hearts are enriched for second heart field (i.e., Isl-1⁺) precursors that give rise to both myocardial and vascular tissues, and may be an appropriate source of progenitor cells for autologous cell-therapy post-MI.     

Systemic Preconditioning by a Prolyl Hydroxylase Inhibitor Promotes Prevention of Skin Flap Necrosis via HIF-1-Induced Bone Marrow-Derived Cells

Background

Local skin flaps often present with flap necrosis caused by critical disruption of the blood supply. Although animal studies demonstrate enhanced angiogenesis in ischemic tissue, no strategy for clinical application of this phenomenon has yet been defined. Hypoxia-inducible factor 1 (HIF-1) plays a pivotal role in ischemic vascular responses, and its expression is induced by the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG). We assessed whether preoperative stabilization of HIF-1 by systemic introduction of DMOG improves skin flap survival.

Methods and Results

Mice with ischemic skin flaps on the dorsum were treated intraperitoneally with DMOG 48 hr prior to surgery. The surviving area with neovascularization of the ischemic flaps was significantly greater in the DMOG-treated mice. Significantly fewer apoptotic cells were present in the ischemic flaps of DMOG-treated mice. Interestingly, marked increases in circulating endothelial progenitor cells (EPCs) and bone marrow proliferative progenitor cells were observed within 48 hr after DMOG treatment. Furthermore, heterozygous HIF-1α-deficient mice exhibited smaller surviving flap areas, fewer circulating EPCs, and larger numbers of apoptotic cells than did wild-type mice, while DMOG pretreatment of the mutant mice completely restored these parameters. Finally, reconstitution of wild-type mice with the heterozygous deficient bone marrow cells significantly decreased skin flap survival.

Conclusion

We demonstrated that transient activation of the HIF signaling pathway by a single systemic DMOG treatment upregulates not only anti-apoptotic pathways but also enhances neovascularization with concomitant increase in the numbers of bone marrow-derived progenitor cells.     

Stem Cell Therapy with Overexpressed VEGF and PDGF Genes Improves Cardiac Function in a Rat Infarct Model

Background

Therapeutic potential was evaluated in a rat model of myocardial infarction using nanofiber-expanded human cord blood derived hematopoietic stem cells (CD133+/CD34+) genetically modified with VEGF plus PDGF genes (VIP).

Methods and Findings

Myocardial function was monitored every two weeks up to six weeks after therapy. Echocardiography revealed time dependent improvement of left ventricular function evaluated by M-mode, fractional shortening, anterior wall tissue velocity, wall motion score index, strain and strain rate in animals treated with VEGF plus PDGF overexpressed stem cells (VIP) compared to nanofiber expanded cells (Exp), freshly isolated cells (FCB) or media control (Media). Improvement observed was as follows: VIP>Exp> FCB>media. Similar trend was noticed in the exercise capacity of rats on a treadmill. These findings correlated with significantly increased neovascularization in ischemic tissue and markedly reduced infarct area in animals in the VIP group. Stem cells in addition to their usual homing sites such as lung, spleen, bone marrow and liver, also migrated to sites of myocardial ischemia. The improvement of cardiac function correlated with expression of heart tissue connexin 43, a gap junctional protein, and heart tissue angiogenesis related protein molecules like VEGF, pNOS3, NOS2 and GSK3. There was no evidence of upregulation in the molecules of oncogenic potential in genetically modified or other stem cell therapy groups.

Conclusion

Regenerative therapy using nanofiber-expanded hematopoietic stem cells with overexpression of VEGF and PDGF has a favorable impact on the improvement of rat myocardial function accompanied by upregulation of tissue connexin 43 and pro-angiogenic molecules after infarction.     

A Plant-Derived Remedy for Repair of Infarcted Heart

Background

Myocardial infarction (MI) due to coronary artery disease remains one of the leading causes of premature death. Replacement of infarcted heart tissue with regenerating myocardium from endogenous progenitor pools or exogenously introduced stem cells remains a therapeutic ideal. Their impracticality mainly lies in their low efficiency in cardiogenic differentiation (CD). Our recent studies with an acute MI animal model have already demonstrated the therapeutic effect of the MeOH extract of Geum japonicum (EGJ), providing clear evidence of myocardial regeneration.

Methods and Findings

The present study further isolated the active component contained in EGJ using bioassay-guided isolation and investigated its efficacy in the treatment of infarcted heart in animal MI models. We demonstrated that substantial repair of infarcted heart in animal MI models by EGJ can be mimicked by the isolated candidate compound (cardiogenin) in MI animal models. Clear evidence of newly regenerated endogenous mesenchymal stem cells (MSCs) derived cardiomyocytes was observed throughout the infarct zone, accompanied by significantly improved functional performance of the heart. Transplantation of MSCs pretreated with EGJ or cardiogenin into a MI animal model also resulted in substantial regeneration of functional myocardium, implying that the activated MSCs carry all the necessary blueprints for myocardial regeneration. Signaling pathways specific to cell survival, CD identified in embryonic heart induction and angiogenesis were activated in both cardiogenin-treated MSCs and cardiogenin-induced regenerating myocardium.

Conclusions

This study has demonstrated the therapeutic effects of cardiogenin in infarcted heart repair, and identified the associated signalling pathways for effective cardiogenic differentiation of MSCs, cell survival and angiogenesis. These findings should enable new treatment strategies for MI to be developed immediately.     

A Survey of the FDA's AERS Database Regarding Muscle and Tendon Adverse Events Linked to the Statin Drug Class

Background

Cholesterol management drugs known as statins are widely used and often well tolerated; however, a variety of muscle-related side effects can arise. These adverse events (AEs) can have serious impact, and form a significant barrier to therapy adherence. Surveillance of post-marketing AEs is of vital importance to understand real-world AEs and reporting differences between individual statin drugs. We conducted a review of post-approval muscle and tendon AE reports in association with statin use, to assess differences within the drug class.

Methods

We analyzed all case reports from the FDA AE Reporting System (AERS) database linking muscle-related AEs to statin use (07/01/2005–03/31/2011). Drugs examined were: atorvastatin, simvastatin, lovastatin, pravastatin, rosuvastatin, and fluvastatin.

Results

Relative risk rates for rosuvastatin were consistently higher than other statins. Atorvastatin and simvastatin showed intermediate risks, while pravastatin and lovastatin appeared to have the lowest risk rates. Relative risk of muscle-related AEs, therefore, approximately tracked with per milligram LDL-lowering potency, with fluvastatin an apparent exception. Incorporating all muscle categories, rates for atorvastatin, simvastatin, pravastatin, and lovastatin were, respectively, 55%, 26%, 17%, and 7.5% as high, as rosuvastatin, approximately tracking per milligram potency (Rosuvastatin>Atorvastatin>Simvastatin>Pravastatin≈Lovastatin) and comporting with findings of other studies. Relative potency, therefore, appears to be a fundamental predictor of muscle-related AE risk, with fluvastatin, the least potent statin, an apparent exception (risk 74% vs rosuvastatin).

Interpretation

AE reporting rates differed strikingly for drugs within the statin class, with relative reporting aligning substantially with potency. The data presented in this report offer important reference points for the selection of statins for cholesterol management in general and, especially, for the rechallenge of patients who have experienced muscle-related AEs (for whom agents of lower expected potency should be preferred).     

Novel Cell-Free Strategy for Therapeutic Angiogenesis: In Vitro Generated Conditioned Medium Can Replace Progenitor Cell Transplantation

Background

Current evidence suggests that endothelial progenitor cells (EPC) contribute to ischemic tissue repair by both secretion of paracrine factors and incorporation into developing vessels. We tested the hypothesis that cell-free administration of paracrine factors secreted by cultured EPC may achieve an angiogenic effect equivalent to cell therapy.

Methodology/Principal Findings

EPC-derived conditioned medium (EPC-CM) was obtained from culture expanded EPC subjected to 72 hours of hypoxia. In vitro, EPC-CM significantly inhibited apoptosis of mature endothelial cells and promoted angiogenesis in a rat aortic ring assay. The therapeutic potential of EPC-CM as compared to EPC transplantation was evaluated in a rat model of chronic hindlimb ischemia. Serial intramuscular injections of EPC-CM and EPC both significantly increased hindlimb blood flow assessed by laser Doppler (81.2±2.9% and 83.7±3.0% vs. 53.5±2.4% of normal, P<0.01) and improved muscle performance. A significantly increased capillary density (1.62±0.03 and 1.68±0.05/muscle fiber, P<0.05), enhanced vascular maturation (8.6±0.3 and 8.1±0.4/HPF, P<0.05) and muscle viability corroborated the findings of improved hindlimb perfusion and muscle function. Furthermore, EPC-CM transplantation stimulated the mobilization of bone marrow (BM)-derived EPC compared to control (678.7±44.1 vs. 340.0±29.1 CD34⁺/CD45⁻ cells/1×10⁵ mononuclear cells, P<0.05) and their recruitment to the ischemic muscles (5.9±0.7 vs. 2.6±0.4 CD34⁺ cells/HPF, P<0.001) 3 days after the last injection.

Conclusions/Significance

Intramuscular injection of EPC-CM is as effective as cell transplantation for promoting tissue revascularization and functional recovery. Owing to the technical and practical limitations of cell therapy, cell free conditioned media may represent a potent alternative for therapeutic angiogenesis in ischemic cardiovascular diseases.