Allison Hanley

Intramyocardial Transplantation of Autologous Endothelial Progenitor Cells for Therapeutic Neovascularization of Myocardial Ischemia

Background—We investigated whether catheter-based, intramyocardial transplantation of autologous endothelial progenitor cells can enhance neovascularization in myocardial ischemia. Methods and Results—Myocardial ischemia was induced by placement of an ameroid constrictor around swine left circumflex artery. Four weeks after constrictor placement, CD31+ mononuclear cells (MNCs) were freshly isolated from the peripheral blood of each animal. After overnight incubation of CD31+ MNCs in noncoated plates, nonadhesive cells (NA/CD31+ MNCs) were harvested as the endothelial progenitor cell–enriched fraction. Nonadhesive CD31− cells (NA/CD31− MNCs) were also prepared. Autologous transplantation of 107 NA/CD31+ MNCs, 107 NA/CD31− MNCs, or PBS was performed with a NOGA mapping injection catheter to target ischemic myocardium. In a parallel study, 105 human CD34+ MNCs, 105 human CD34− MNCs, or PBS was transplanted into ischemic myocardium of nude rats 10 minutes after ligation of the left anterior descending coronary artery. In the swine study, ischemic area by NOGA mapping, Rentrop grade angiographic collateral development, and echocardiographic left ventricular ejection fraction improved significantly 4 weeks after transplantation of NA/CD31+ MNCs but not after injection of NA/CD31− MNCs or PBS. Capillary density in ischemic myocardium 4 weeks after transplantation was significantly greater in the NA/CD31+ MNC group than the control groups. In the rat study, echocardiographic left ventricular systolic function and capillary density were significantly better preserved in the CD34+ MNC group than in the control groups 4 weeks after myocardial ischemia. Conclusions—These favorable outcomes encourage future clinical trials of catheter-based, intramyocardial transplantation of autologous CD34+ MNCs in the setting of chronic myocardial ischemia.

Clonally expanded novel multipotent stem cells from human bone marrow regenerate myocardium after myocardial infarction

We have identified a subpopulation of stem cells within adult human BM, isolated at the single-cell level, that self-renew without loss of multipotency for more than 140 population doublings and exhibit the capacity for differentiation into cells of all 3 germ layers. Based on surface marker expression, these clonally expanded human BM-derived multipotent stem cells (hBMSCs) do not appear to belong to any previously described BM-derived stem cell population. Intramyocardial transplantation of hBMSCs after myocardial infarction resulted in robust engraftment of transplanted cells, which exhibited colocalization with markers of cardiomyocyte (CMC), EC, and smooth muscle cell (SMC) identity, consistent with differentiation of hBMSCs into multiple lineages in vivo. Furthermore, upregulation of paracrine factors including angiogenic cytokines and antiapoptotic factors, and proliferation of host ECs and CMCs, were observed in the hBMSC-transplanted hearts. Coculture of hBMSCs with CMCs, ECs, or SMCs revealed that phenotypic changes of hBMSCs result from both differentiation and fusion. Collectively, the favorable effect of hBMSC transplantation after myocardial infarction appears to be due to augmentation of proliferation and preservation of host myocardial tissues as well as differentiation of hBMSCs for tissue regeneration and repair. To our knowledge, this is the first demonstration that a specific population of multipotent human BM-derived stem cells can induce both therapeutic neovascularization and endogenous and exogenous cardiomyogenesis.

Estrogen-Mediated, Endothelial Nitric Oxide Synthase–Dependent Mobilization of Bone Marrow–Derived Endothelial Progenitor Cells Contributes to Reendothelialization After Arterial Injury

Background—We hypothesized that estrogen-induced acceleration of reendothelialization might be mediated in part by effects involving mobilization and incorporation of bone marrow–derived endothelial progenitor cells (EPCs). Methods and Results—Carotid injury was induced in ovariectomized wild-type mice receiving either 17&bgr;-estradiol or placebo. Estradiol treatment significantly accelerated reendothelialization of injured arterial segments within 7 days and resulted in a significant reduction of medial thickness 14 and 21 days after the injury. Significant increases in circulating EPCs 3 days after the injury were observed in the estradiol group compared with placebo-treated mice. These data were further supported by fluorescence-activated cell sorting analysis, which disclosed a significant increase in Sca-1/Flk-1–positive cells in estradiol versus control mice. To evaluate the effects of estradiol on bone marrow–derived EPC incorporation at sites of reendothelialization, carotid injury was established in ovariectomized wild-type mice transplanted with bone marrow from transgenic donors expressing &bgr;-galactosidase transcriptionally regulated by the Tie-2 promoter. Significantly greater numbers of X-gal–positive cells were observed at reendothelialized areas in the estradiol group 3 days after injury as compared with placebo. Fluorescent immunohistochemistry 14 days after the injury documented a marked increase in cells expressing both &bgr;-gal, indicating bone marrow origin and Tie-2 expression, and isolectin B4, also indicating endothelial lineage, in the estradiol group compared with control. In contrast, estradiol did not accelerate reendothelialization or augment EPC mobilization into the peripheral circulation after injury in endothelial nitric oxide synthase–deficient mice (eNOS−/−). Furthermore, estradiol exhibited direct stimulatory effects on EPC mitogenic and migration activity and inhibited EPC apoptosis. Conclusions—Estradiol accelerates reendothelialization and attenuates medial thickening after carotid injury in part by augmenting mobilization and proliferation of bone marrow–derived EPCs and their incorporation into the recovering endothelium at the site of injury.

CD34-Positive Cells Exhibit Increased Potency and Safety for Therapeutic Neovascularization After Myocardial Infarction Compared With Total Mononuclear Cells

Background— We compared the therapeutic potential of purified mobilized human CD34+ cells with that of mobilized total mononuclear cells (tMNCs) for the preservation/recovery of myocardial tissue integrity and function after myocardial infarction (MI). Methods and Results— CD34+ cells were purified from peripheral blood tMNCs of healthy volunteers by magnetic cell sorting after a 5-day administration of granulocyte colony-stimulating factor. Phosphate-buffered saline (PBS), 5×105 CD34+ cells/kg, 5×105 tMNCs/kg (low-dose MNCs [loMNCs]), or a higher dose of tMNCs (hiMNCs) containing 5×105 CD34+ cells/kg was transplanted intramyocardially 10 minutes after the induction of MI in athymic nude rats. Hematoxylin and eosin staining revealed that moderate to severe hemorrhagic MI on day 3 was more frequent in the hiMNC group than in the PBS and CD34+ cell groups. Immunostaining for human-specific CD45 revealed abundant distribution of hematopoietic/inflammatory cells derived from transplanted cells in the ischemic myocardium of the hiMNC group. Capillary density on day 28 was significantly greater in the CD34+ cell group (721.1±19.9 per 1 mm2) than in the PBS, loMNC, and hiMNC groups (384.7±11.0, 372.5±14.1, and 497.5±24.0 per 1 mm2) (P<0.01). Percent fibrosis area on day 28 was less in the CD34+ cell group (15.6±0.9%) than in the PBS, loMNC, and hiMNC groups (26.3±1.2%, 27.5±1.8%, and 22.2±1.8%) (P<0.05). Echocardiographic fractional shortening on day 28 was significantly higher in the CD34+ cell group (30.3±0.9%) than in the PBS, loMNC, and hiMNC groups (22.7±1.5%, 23.4±1.1%, and 24.9±1.7%; P<0.05). Echocardiographic regional wall motion score was better preserved in the CD34+ cell group (21.8±0.5) than in the PBS, loMNC, and hiMNC groups (25.4±0.4, 24.9±0.4, and 24.1±0.6; P<0.05). Conclusions— CD34+ cells exhibit superior efficacy for preserving myocardial integrity and function after MI than unselected circulating MNCs.

Progressive Attenuation of Myocardial Vascular Endothelial Growth Factor Expression Is a Seminal Event in Diabetic Cardiomyopathy Restoration of Microvascular Homeostasis and Recovery of Cardiac Function in Diabetic Cardiomyopathy After Replenishment of Local Vascular Endothelial Growth Factor

Background—Diabetic cardiomyopathy (DCM) is characterized by microvascular pathology and interstitial fibrosis, which leads to progressive heart failure; however, the pathogenesis of DCM remains uncertain. Methods and Results—Using the streptozotocin-induced diabetic rat model, we evaluated the natural course of DCM over a period of 1 year by serial echocardiography, Western blot analysis for vascular endothelial growth factor (VEGF), endothelial progenitor cell assays, myocardial blood flow measurements, and histopathologic analysis that included terminal dUTP nick end-labeling (TUNEL), capillary and cardiomyocyte density, and fibrosis area. Downregulation of myocardial VEGF expression preceded all other features of DCM and was followed by increased apoptosis of endothelial cells, decreased numbers of circulating endothelial progenitor cells, decreased capillary density, and impaired myocardial perfusion. Apoptosis and necrosis of cardiomyocytes ensued, along with fibrosis and progressive diastolic and then systolic dysfunction. To provide further evidence of the central role of VEGF in the pathophysiology of DCM, we replenished myocardial VEGF expression using naked DNA gene therapy via direct intramyocardial injection of plasmid DNA encoding VEGF (phVEGF165). VEGF-replenished rats showed increased capillary density, decreased endothelial cell and cardiomyocyte apoptosis, and in situ differentiation of bone marrow–derived endothelial progenitor cells into endothelial cells. These anatomic findings were accompanied by significant improvements in cardiac function. Conclusions—These findings suggest that downregulation of VEGF may compromise microvascular homeostasis in the myocardium and thereby play a central role in the pathogenesis of DCM.

Local Gene Transfer of phVEGF-2 Plasmid by Gene-Eluting Stents An Alternative Strategy for Inhibition of Restenosis

Background—Drug-eluting stents represent a useful strategy for the prevention of restenosis using various antiproliferative drugs. These strategies share the liability of impairing endothelial recovery, thereby altering the natural biology of the vessel wall and increasing the associated risk of stent thrombosis. Accordingly, we tested the hypothesis that local delivery via gene-eluting stent of naked plasmid DNA encoding for human vascular endothelial growth factor (VEGF)-2 could achieve similar reductions in neointima formation while accelerating, rather than inhibiting, reendothelialization. Methods and Results—phVEGF 2-plasmid (100 or 200 μg per stent)–coated BiodivYsio phosphorylcholine polymer stents versus uncoated stents were deployed in a randomized, blinded fashion in iliac arteries of 40 normocholesterolemic and 16 hypercholesterolemic rabbits. Reendothelialization was nearly complete in the VEGF stent group after 10 days and was significantly greater than in control stents (98.7±1% versus 79.0±6%, P <0.01). At 3 months, intravascular ultrasound analysis revealed that lumen cross-sectional area (4.2±0.4 versus 2.27±0.3 mm2, P <0.001) was significantly greater and percent cross-sectional narrowing was significantly lower (23.4±6 versus 51.2±10, P <0.001) in VEGF stents compared with control stents implanted in hypercholesterolemic rabbits. Transgene expression was detectable in the vessel wall along with improved functional recovery of stented segments, resulting in a 2.4-fold increase in NO production. Conclusions—Acceleration of reendothelialization via VEGF-2 gene–eluting stents provides an alternative treatment strategy for the prevention of restenosis. VEGF-2 gene–eluting stents may be considered as a stand-alone or combination therapy.

Synergistic effect of bone marrow mobilization and vascular endothelial growth factor-2 gene therapy in myocardial ischemia.

Background—We performed a series of investigations to test the hypothesis that combining angiogenic gene therapy and cytokine (CK)-induced endothelial progenitor cell mobilization would be superior to either strategy alone for treatment of chronic myocardial ischemia. Methods and Results—A swine model of chronic myocardial ischemia and a murine model of acute myocardial infarction were used in this study. In both models, animals were randomly assigned to 1 of 4 treatment groups: Combo group, intramyocardial vascular endothelial growth factor (VEGF)-2 gene transfer plus subcutaneous injection of CKs; VEGF-2, VEGF-2 gene transfer plus saline subcutaneously injected; CK, empty vector transfer plus CKs; and control, empty vector plus subcutaneous saline. Acute myocardial infarction was also induced in wild-type mice 4 weeks after bone marrow transplantation from enhanced green fluorescent protein transgenic mice to permit observation of bone marrow–derived cells in the myocardium after acute myocardial infarction. In chronic myocardial ischemia, combination therapy resulted in superior improvement in all indexes of perfusion and function compared with all other treatment groups. In the bone marrow transplant mice, double immunofluorescent staining revealed that the combination of CK-induced mobilization and local VEGF-2 gene transfer resulted in a significant increase in the number of bone marrow–derived cells incorporating into the neovasculature, indicating that recruitment and/or retention of bone marrow–derived progenitors was enhanced by mobilization and that local VEGF-2 gene transfer can provide signals for recruitment or incorporation of circulating progenitor cells. Conclusions—Mobilization of endothelial progenitor cells with cytokines potentiates VEGF-2 gene therapy for myocardial ischemia and enhances bone marrow cell incorporation into ischemic myocardium.

The Neuropeptide Secretoneurin Acts as a Direct Angiogenic Cytokine In Vitro and In Vivo

Background—Secretoneurin is an abundant neuropeptide of the central, peripheral, and autonomic nervous systems, located in nerve fibers characterized by a close interaction with blood vessels and known to stimulate endothelial cell migration. Methods and Results—We hypothesized that secretoneurin might act as an angiogenic cytokine and tested for these effects in vivo using a mouse cornea neovascularization model and in vitro by assessing capillary tube formation in a matrigel assay. In vivo, secretoneurin-induced neovasculature is characterized by a distinct pattern of arterial and venous vessels of large diameter and length. Immunohistochemical staining for CD-31 revealed endothelial lining of the inner surface of these vessels, and recruitment of &agr;-smooth muscle actin–positive perivascular cells suggests vessel maturation. In vitro, secretoneurin-induced capillary tube formation was dose dependent and specific, confirming that effects of secretoneurin occur directly on endothelial cells. Secretoneurin also stimulated proliferation and exerted antiapoptotic effects on endothelial cells and activated intracellular phosphatidylinositol 3′ kinase/Akt and mitogen-activated protein kinase pathways, as demonstrated by increased phosphorylation of Akt and extracellular signal–regulated kinase. Conclusions—These data show that secretoneurin represents a novel direct angiogenic cytokine and reiterate the coordinated relationship between nervous and vascular systems.

The cytoskeletal protein ezrin regulates EC proliferation and angiogenesis via TNF-α-induced transcriptional repression of cyclin A

TNF-alpha modulates EC proliferation and thereby plays a central role in new blood vessel formation in physiologic and pathologic circumstances. TNF-alpha is known to downregulate cyclin A, a key cell cycle regulatory protein, but little else is known about how TNF-alpha modulates EC cell cycle and angiogenesis. Using primary ECs, we show that ezrin, previously considered to act primarily as a cytoskeletal protein and in cytoplasmic signaling, is a TNF-alpha-induced transcriptional repressor. TNF-alpha exposure leads to Rho kinase-mediated phosphorylation of ezrin, which translocates to the nucleus and binds to cell cycle homology region repressor elements within the cyclin A promoter. Overexpression of dominant-negative ezrin blocks TNF-alpha-induced modulation of ezrin function and rescues cyclin A expression and EC proliferation. In vivo, blockade of ezrin leads to enhanced transplanted EC proliferation and angiogenesis in a mouse hind limb ischemia model. These observations suggest that TNF-alpha regulates angiogenesis via Rho kinase induction of a transcriptional repressor function of the cytoskeletal protein ezrin and that ezrin may represent a suitable therapeutic target for processes dependent on EC proliferation.

Secretoneurin, an Angiogenic Neuropeptide, Induces Postnatal Vasculogenesis

Background—Induction of postnatal vasculogenesis, the mobilization of bone marrow–derived endothelial progenitor cells and incorporation of these cells into sites of blood vessel formation, is a well-known feature of angiogenic cytokines such as vascular endothelial growth factor. We hypothesized that the angiogenic neuropeptide secretoneurin induces this kind of neovascularization. Methods and Results—Secretoneurin induced mobilization of endothelial progenitor cells to sites of vasculogenesis in vivo in the cornea neovascularization assay. Progenitor cells were incorporated into vascular structures or were located adjacent to them. Systemic injection of secretoneurin led to increase of circulating stem cells and endothelial progenitor cells. In vitro secretoneurin induced migration, exerted antiapoptotic effects, and increased the number of these cells. Furthermore, secretoneurin stimulated the mitogen-activated protein kinase system, as shown by phosphorylation of extracellular signal–regulated kinase, and activated the protein kinase B/Akt pathway. Activation of mitogen-activated protein kinase was necessary for increase of cell number and migration, whereas Akt seemed to play a role in migration of endothelial progenitor cells. Conclusions—These data show that the angiogenic neuropeptide secretoneurin stimulates postnatal vasculogenesis by mobilization, migration, and incorporation of endothelial progenitor cells.