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Background— Toxic neuropathies induced by cisplatin and other chemotherapeutic agents are important clinical problems because of their high incidence, their lack of effective treatment, and the fact that neuropathy represents a dose-limiting factor for these therapies. The pathogenic basis for toxic neuropathies induced by chemotherapeutic agents has not been completely elucidated. Methods and Results— We investigated the hypothesis that experimental toxic neuropathy results from an antiangiogenic effect of these drugs, resulting in destruction of the vasa nervorum, and accordingly that the neuropathy could be prevented or reversed by locally administered VEGF gene transfer without augmenting tumor growth. In an animal model of cisplatin-induced neuropathy, nerve blood flow was markedly attenuated, and there was a profound reduction in the number of vasa nervorum associated with marked endothelial cell apoptosis, resulting in a severe peripheral neuropathy with focal axonal degeneration characteristic of ischemic neuropathy. After intramuscular gene transfer of naked plasmid DNA encoding VEGF-1 in animals with an established neuropathy, vascularity and blood flow returned to levels similar to those of control rats, peripheral nerve function was restored, and histological nerve architecture was normalized. Gene therapy administered in parallel with cisplatin chemotherapy completely attenuated endothelial cell apoptosis and inhibited destruction of nerve vasculature, deterioration of nerve function, and axonal degeneration. In a rat tumor model, VEGF gene transfer administered locally did not alter tumor growth or vascularity. Conclusions— These findings implicate microvascular damage as the basis for toxic neuropathy induced by cisplatin and suggest that local angiogenic gene therapy may constitute a novel prevention or treatment for this disorder without augmenting tumor growth or vascularization.

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 (phVEGF 165 ). 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.

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.

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 2 , P P 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.

Several recent studies have demonstrated the potential for improving myocardial perfusion by the continuous administration of angiogenic growth factors. Studies in our laboratory have shown that a single intraarterial or intravenous bolus of the endothelial cell specific mitogen vascular endothelial growth factor (VEGF) can significantly improve perfusion in a rabbit ischemic limb model. To test the efficacy of this therapeutic approach in chronic myocardial ischemia, 18 Yorkshire pigs underwent a left thoracotomy followed by placement of an ameroid constrictor around the proximal circumflex coronary artery. Gradual occlusion of the artery (26 +/- 4 days) was accompanied by identifiable hypokinesis of the posterolateral wall of the left ventricle (2D echo). Thirty days postoperatively, rhVEGF(165) (2 mg; n = 8) or saline (n = 10) was administered directly into the left coronary ostium. Postadenosine myocardial perfusion studies using colored microspheres 30 days later demonstrated superior blood flow in the ischemic zone of the VEGF-treated hearts (ischemic/normal ratio 1.09 vs 0.97, P0.05) compared with those receiving saline injection. Four of eight VEGF-treated animals succumbed, however, to severe hypotension following VEGF administration. Therefore 500 micrograms of VEGF were administered intracoronary to five normal pigs. A significant drop in mean arterial pressure (-44.4 +/- 3.2%, P0.05 vs baseline) and peripheral resistance (-13.2 +/- 4.5%, P0.05 vs baseline) was accompanied by increased heart rate. IV administration of N(omega)-nitro-L-arginine (L-NNA), an EDRF inhibitor, restored blood pressure to baseline. We conclude that a single intracoronary bolus of VEGF is capable of significantly augmenting flow to collateral-dependent ischemic myocardium. The associated hypotension appears to be EDRF-mediated. Further studies are needed to define the best dose and route of administration of VEGF for the treatment of coronary insufficiency.

Toxic neuropathy represents an important clinical problem in the use of the chemotherapeutic substances Taxol and thalidomide. Sensory neuropathy has a high incidence, lacks an effective treatment and is the dose-limiting factor for these drugs. The pathogenic basis of these neuropathies is unknown. We investigated the hypothesis that the experimental toxic neuropathies from Taxol and thalidomide results from destruction of vasa nervorum and can be reversed by the administration of an angiogenic cytokine. In animal models of Taxol- and thalidomide-induced neuropathy, nerve blood flow has been attenuated and the number of vasa nervorum has been reduced. Intramuscular gene transfer of naked plasmid DNA encoding VEGF-1 administered in parallel with Taxol injections completely inhibited deterioration of nerve function and diminution of the peripheral nerve vasculature. Gene therapy in animals with established Taxol- or thalidomide-induced neuropathies resulted in recovery of vascularity and improved nerve electrophysiology. These findings implicate microvascular damage as the basis for toxic neuropathy and suggest that angiogenic growth factors may constitute a novel treatment for this disorder.

Background — Primary and secondary prevention trials suggest that statins possess favorable effects independent of cholesterol reduction. We investigated whether statin therapy may also accelerate reendothelialization after carotid balloon injury. Methods and Results — Simvastatin treatment in 34 male Sprague-Dawley rats accelerated reendothelialization of the balloon-injured arterial segments (reendothelialized area at 2 weeks, 12.3±1.8 versus 5.4±1.1 mm 2 , P < 0.01) and resulted in a dose-dependent (0.2 or 1 mg/kg IP) significant reduction in neointimal thickening at 2, 3, and 4 weeks compared with saline-injected controls (n=18). To elucidate the mechanism, we investigated the contribution of bone marrow–derived endothelial progenitor cells (EPCs) by bone marrow transplantation from Tie2/lacZ mice to background mice or nude rats. X-gal staining of mouse carotid artery specimens revealed a 2.9-fold increase in the number of β-gal–positive cells per square millimeter appearing on the carotid artery luminal surface at 2 weeks, and double-fluorescence immunohistochemistry disclosed a significant 5-fold increase in the number of double-positive cells (β-gal, isolectin B4) on the luminal surface in carotid arteries of statin-treated nude rats (20±3 versus 4±1 cells/mm surface length, P P 5 , β 1 , α v , and β 5 of human EPCs as shown by reverse transcription–polymerase chain reaction and fluorescence-activated cell sorting. Conclusions — These findings establish additional mechanisms by which statins may specifically preempt disordered vascular wall pathology and constitute physiological evidence that EPC mobilization represents a functionally relevant consequence of statin therapy.