Therapeutic angiogenesis inhibits or rescues chemotherapy-induced peripheral neuropathy: taxol- and thalidomide-induced injury of vasa nervorum is ameliorated by VEGF.

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.

Intramuscular gene transfer of fibroblast growth factor-1 using improved pCOR plasmid design stimulates collateral formation in a rabbit ischemic hindlimb model.

Fibroblast growth factor 1 (FGF1) is an angiogenic factor known to play a role in the growth of arteries. The purpose of this study was to evaluate the usefulness of direct intramuscular injection of an optimized expression plasmid encoding FGF1 to augment collateral formation and tissue perfusion in a rabbit ischemic hindlimb model. Truncated FGF1 fused to the human fibroblast interferon (FIN) signal peptide was expressed from a newly designed plasmid backbone with an improved safety profile for gene therapy applications. In vitro, optimization of plasmid design yielded in a dramatic increase in expression efficiency for FGF1, independent of the presence of a signal peptide, as analyzed by Western Blotting. In vivo, successful transgene expression could be demonstrated by FGF1 immunostaining after gene application. FGF1 plasmid containing FIN signal peptide (100, 500, and 1,000 mug), when injected into ischemic muscle areas of rabbits 10 days after ligation of the external iliac artery, exhibited a pronounced therapeutic effect on collateral formation to the ischemic hindlimb in a dose-depending manner, as assessed by physiological (blood pressure ratio, maximal intra-arterial Doppler flow) and anatomical (angiographic score, histologic evaluation of capillary density) measurements 30 days after therapy, compared to saline or lacZ control plasmid. FGF1 plasmid without a signal peptide sequence resulted in a comparable therapeutic effect on collateral formation at comparable doses (500 and 1,000 mug). Our results indicate that intramuscular FGF1 gene application could be useful to stimulate collateral formation in a situation of chronic peripheral ischemia. The presence of a signal peptide does not seem to be obligatory to achieve bioactivity of intramuscular transfected FGF1. An optimized vector design improved both biosafety of gene transfer and expression efficiency of the transgene, rendering this vector highly suitable for human gene therapy. Therefore, this new generation vector encoding FGF1 might be useful as an alternative treatment for patients with chronic ischemic disorders not amenable to conventional therapy.

Hyperhomocyst(e)inemia impairs angiogenesis in a murine model of limb ischemia.

Hyperhomocyst(e)inemia (HH) is an established independent risk factor for coronary, cerebral and peripheral vascular diseases. Recent studies have indicated that certain cardiovascular risk factors, including diabetes and hypercholesterolemia, impair expression of vascular endothelial growth factor (VEGF) and endogenous angiogenesis. In this study, we investigate the impact of moderate HH on angiogenesis and VEGF pathway in a mouse model of hindlimb ischemia. Upon induction of unilateral hindlimb ischemia, endogenous angiogenesis, expression of VEGF, and phosphorylation of the VEGF receptor Flk-1 were evaluated in mice heterozygous for a deletion of the cystathionine beta-synthase gene (CBS) and compared with those observed in CBS+/+ mice. CBS+/- mice exhibit moderate HH, as demonstrated by measuring plasma total homocyst(e)ine (tHcy) levels, which were significantly higher in these animals compared with CBS+/+ mice (4.77 +/- 0.82 vs 2.10 +/- 0.28, p < 0.01). Twenty-eight days after induction of ischemia, hindlimb blood flow was significantly reduced in CBS+/- mice compared with CBS+/+ animals (0.49 +/- 0.03, n = 12 vs 0.71 +/- 0.09, n = 10; p < 0.05). In addition, there was a significant negative correlation between plasma homocyst(e)ine levels and the laser Doppler perfusion ratio in CBS+/- mice (p = 0.0087, r = -0.7171). While VEGF expression and Flk-1 phosphorylation were not impaired in the ischemic muscles of CBS+/- mice, phosphorylation of the endothelial cell survival factor Akt was significantly inhibited by homocyst(e)ine in a dose-dependent manner in human umbilical vein endothelial cell (HUVECs) in vitro. In conclusion, our findings demonstrate that endogenous angiogenesis is inversely related to plasma levels of homocyst(e)ine in genetically engineered, heterozygous mice with moderate HH. This impairment, however, is not dependent on reduced expression of VEGF or impaired phosphorylation of its receptor Flk-1. In contrast, our data suggest that impaired Akt phosphorylation mediates the impairment of angiogenesis associated with HH.

Antiangiogenesis mediates cisplatin-induced peripheral neuropathy: attenuation or reversal by local vascular endothelial growth factor gene therapy without augmenting tumor growth.

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.

Functional ephrin-B2 expression for promotive interaction between arterial and venous vessels in postnatal neovascularization.

BACKGROUND: Ephrin-B2, one of the transmembrane ligands, is a genetic marker of arterial endothelial cells (ECs) at embryonic stages and is essential for cardiovascular development, but its roles in ischemic cardiovascular disease are not well understood. In this study, we focused on the function of ephrin-B2 in postnatal neovascularization. METHODS AND RESULTS: We found that ephrin-B2 is exclusively expressed and significantly upregulated in the arterial vasculature after the initial angiogenic responses in tissue ischemia. Upregulation of ephrin-B2 is also observed in EC cordlike formation in vitro. Interestingly, ephrin-B2 expression on ECs was enhanced by promotive angiogenic growth factors, such as vascular endothelial growth factor, basic fibroblast growth factor, and hepatocyte growth factor, whereas it was attenuated by angiopoietin-1, a factor for blood vessel maturation. Moreover, an ephrin-B2-rich environment was shown to induce neovascularization mainly through venous angiogenesis in an in vivo cornea micropocket assay. CONCLUSIONS: Our study indicates that the ephrin-B2 ligand is likely to have functional expression on angiogenic arterial ECs and induce a subsequent promotive effect on venous vessels during postnatal neovascularization.

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.

Long-term (2-year) clinical events following transthoracic intramyocardial gene transfer of VEGF-2 in no-option patients.

BACKGROUND: The short-term clinical impact of intramyocardial gene transfer (GT) of the angiogenic protein vascular endothelial growth factor-2 (VEGF-2) has been previously reported to significantly reduce Canadian Cardiovascular Society (CCS) angina class and to prolong exercise treadmill test (ETT) time. We describe the safety and long-term events (>1 year) in consecutive, nonrandomized, patients who received intramyocardial VEGF-2. METHODS: Thirty patients with intractable CCS class III or IV angina and no options for revascularization underwent direct intramyocardial GT of VEGF-2 naked DNA via limited thoracotomy at total doses of 0.2, 0.8, or 2.0 mg. Patients were followed for clinical events after 1 year by hospital records, follow-up visits or telephone contact. Due to one perioperative death, 29 patients were followed. RESULTS: At a mean follow-up of 751 +/- 102.5 days (range 459-959) there were four deaths (13.8%), five myocardial infarctions (MIs) (17.2%), and seven revascularization procedures (24.1%). There were 15 hospitalizations in 12 patients. At the end of the follow-up period no patient (0%) had CCS class IV angina, 3 patients (11.5%) had class III angina, and 23 (88.5%) had class I to II angina. There were two new diagnoses of cancer. CONCLUSION: Transthoracic intramyocardial injection of VEGF-2 is associated with an improvement of symptoms of angina in the majority of patients beyond the first year of treatment. Major clinical events such as death, MI, and repeat revascularization are uncommon during the first year but more frequent after 1 year at a rate consistent with the severity of underlying disease in this population with advanced atherosclerosis. The majority of events were the result of progression of disease in areas of the heart remote from the site of GT. A large randomized trial is planned to determine the efficacy of intramyocardial VEGF-2 injections in inoperable patients.

Age-dependent VEGF expression and intraneural neovascularization during regeneration of peripheral nerves.

The physiologic ability of peripheral nerves to regenerate after injury is impaired with aging. However, the mechanisms responsible for this phenomenon are still incompletely characterized. In this study, we investigated whether aging influences the intraneural angiogenic response that occurs after injury and during regeneration of peripheral nerves. We performed a crush injury of the sciatic nerve in old and senescence accelerated mice and found that the peripheral nerves of these animals are unable to locally upregulate vascular endothelial growth factor (VEGF), a prototypical angiogenic cytokine, after injury and have substantial deficits in mounting an appropriate intraneural angiogenic response during nerve regeneration. Our findings provide new evidence of possible interdependent relationships between aging, VEGF, angiogenesis, and nerve regeneration and suggest that vascular abnormalities might play a role in aging-associated neurological dysfunction, with potentially important fundamental and clinical implications.

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 microg 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 mm(2), 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.

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 17beta-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 beta-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 beta-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.

One-year follow-up of direct myocardial gene transfer of vascular endothelial growth factor-2 using naked plasmid deoxyribonucleic acid by way of thoracotomy in no-option patients.

This phase I open label, dose-escalating study shows that gene transfer of vascular endothelial growth factor-2 naked deoxyribonucleic acid by direct myocardial injection by way of thoracotomy in patients with Canadian Cardiovascular Society class 3 or 4 angina is feasible and safe. The procedure is well tolerated, with few major adverse cardiac events at 1 year, and without complications directly related to gene expression. In this prospective, nonblinded study, the procedure is associated with clinical improvement; however, there was no angiographic evidence of angiogenesis and there is a great potential for a sham or placebo effect in the study patients. A randomized phase III trial is underway that will help determine the efficacy of vascular endothelial growth factor-2 gene transfer in "no-option" patients.

Postnatal recapitulation of embryonic hedgehog pathway in response to skeletal muscle ischemia.

BACKGROUND: Hedgehog (Hh) proteins are morphogens regulating epithelial-mesenchymal signaling during several crucial processes of embryonic development, including muscle patterning. Sonic (Shh), Indian (Ihh), and Desert (Dhh) hedgehog constitute the repertoire of Hh genes in humans. The activities of all 3 are transduced via the Patched (Ptc1) receptor. Recent observations indicate that exogenous administration of Shh induces angiogenesis. Here, we studied whether the endogenous Hh pathway, in addition to its functions during embryogenesis, plays a physiological role in muscle regeneration after ischemia in adults. METHODS AND RESULTS: We found that skeletal muscle ischemia induces strong local upregulation of Shh mRNA and protein. In addition, the Ptc1 receptor is activated in interstitial mesenchymal cells within the ischemic area, indicating that these cells respond to Shh and that the Shh pathway is functional. We also found that Shh-responding cells produce vascular endothelial growth factor under ischemic conditions and that systemic treatment with a Shh-blocking antibody inhibits the local angiogenic response and the upregulation of vascular endothelial growth factor. CONCLUSIONS: Our study shows that the Hh signaling may be recapitulated postnatally in adult and fully differentiated muscular tissues and has a regulatory role on angiogenesis during muscle regeneration after ischemia. These findings demonstrate a novel biological activity for the Hh pathway with both fundamental and potential therapeutic implications.

Engineering the response to vascular injury: divergent effects of deregulated E2F1 expression on vascular smooth muscle cells and endothelial cells result in endothelial recovery and inhibition of neointimal growth.

Tumor necrosis factor-alpha (TNF-alpha) is expressed locally in the vessel wall after angioplasty and induces growth arrest and apoptosis in endothelial cells (ECs), thereby delaying reendothelialization. Prior studies have shown that direct antagonism of TNF-alpha, using a systemically administered soluble receptor, can enhance endothelial recovery and reduce neointimal thickening. These studies have also shown that downregulation of the transcription factor E2F1 was a key mechanism of TNF's effect on ECs. We now show that Ad-E2F1 overexpression at sites of balloon injury accelerates functional endothelial recovery, consistent with the prior in vitro findings. Moreover these studies also reveal divergent effects of TNF-alpha and overexpression of E2F1 on ECs versus VSMCs. TNF-alpha exposure of VSMCs had no affect on proliferation or apoptosis, in contrast to the effect seen in ECs. In Ad-E2F1-transduced VSMCs, however, TNF-alpha-induced marked apoptosis in contrast to the survival effect seen in ECs. Finally, these studies suggest that differential activation of NF-kappaB may play a key role in mediating these opposing effects. Nuclear translocation and transcriptional activity of NF-kappaB was markedly attenuated in Ad-E2F1-transduced VSMCs, whereas it remained active in similarly treated ECs when the cells were exposed to TNF-alpha. These studies reveal that overexpression of Ad-E2F1 primes VSMCs to TNF-alpha-induced apoptosis. Furthermore, E2F1 potentiates VSMC death by blocking antiapoptotic signaling pathway through inhibition of NF-kappaB activation. The divergent responses of VSMCs and ECs to E2F1 overexpression provide unique therapeutic possibilities: simultaneously targeting the cell cycle of two different cell types, within same tissue microenvironment resulting in opposite and biologically complimentary effects.

Synergistic effect of angiopoietin-1 and vascular endothelial growth factor on neoangiogenesis in hypercholesterolemic rabbit model with acute hindlimb ischemia.

Vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang1) are essential for vascular integrity and development. The purpose of the study was to test the hypothesis that Ang1 will promote angiogenic response to VEGF in the spontaneous Watanabe heritable hypercholesterolemic (WHHL) rabbit model of acute hindlimb ischemia. Immediately after the ligation of the external iliac artery and the excision of the common and superficial femoral artery in one female WHHL rabbit, 250 microg of phVEGF(165) (n = 8), 500 microg of pAng1* (n = 8), or 250 microg of phVEGF(165) plus 500 microg of pAng1* (n = 8) was injected intramuscularly into the ischemic hindlimb muscles. Gross appearance of ischemic limb, collateral vessel formation and limb perfusion were assessed 30 days after treatment. The incidence of ischemic limb necrosis was higher in the animals treated by phVEGF(165) or by pAng1* than in those treated by phVEGF(165) plus pAng1* (100%, 75% and 14.3%, respectively; P = 0.002). Animals in the combination therapy group had a significantly higher calf blood pressure ratio at day 30 (VEGF plus Ang1* = 0.84 +/- 0.06; VEGF = 0.54 +/- 0.01; Ang1* = 0.59 +/- 0.05; P < 0.01). A combination therapy of VEGF plus Ang*1 had a significantly higher (P < 0.01) angiographic score than either therapy alone. Capillary density (P < 0.05) and capillary/muscle fiber ratio (P < 0.01) of the combination therapy group were also significantly higher than that of either therapy alone. In conclusion, Ang1 can potentiate the angiogenic response to VEGF in the hyperlipidemic rabbit model of acute hindlimb ischemia. Intramuscular administration of cytokines on revascularization of the ischemic hindlimb model of hyperlipidemic rabbit is feasible.

Stromal cell-derived factor-1 effects on ex vivo expanded endothelial progenitor cell recruitment for ischemic neovascularization.

BACKGROUND: Stromal cell-derived factor-1 (SDF-1) is a chemokine considered to play an important role in the trafficking of hematopoietic stem cells. Given the close relationship between hematopoietic stem cells and endothelial progenitor cells (EPCs), we investigated the effect of SDF-1 on EPC-mediated vasculogenesis. METHODS AND RESULTS: Flow cytometric analysis demonstrated expression of CXCR4, the receptor of SDF-1, by 66+/-3% of EPCs after 7 days in culture. In vitro modified Boyden chamber assay showed a dose-dependent EPC migration toward SDF-1 (control versus 10 ng/mL SDF-1 versus 100 ng/mL SDF-1, 24+/-2 versus 71+/-3 versus 140+/-6 cells/mm2; P<0.0001). SDF-1 attenuated EPC apoptosis (control versus SDF-1, 27+/-1 versus 7+/-1%; P<0.0001). To investigate the effect of SDF-1 in vivo, we locally injected SDF-1 into athymic ischemic hindlimb muscle of nude mice combined with human EPC transplantation to determine whether SDF-1 augmented EPC-induced vasculogenesis. Fluorescence microscopic examination disclosed increased local accumulation of fluorescence-labeled EPCs in ischemic muscle in the SDF-1 treatment group (control versus SDF-1=241+/-25 versus 445+/-24 cells/mm2, P<0.0001). At day 28 after treatment, ischemic tissue perfusion was improved in the SDF-1 group and capillary density was also increased. (control versus SDF-1, 355+/-26 versus 551+/-30 cells/mm2; P<0.0001). CONCLUSION: These findings indicate that locally delivered SDF-1 augments vasculogenesis and subsequently contributes to ischemic neovascularization in vivo by augmenting EPC recruitment in ischemic tissues.

VEGF-C gene therapy augments postnatal lymphangiogenesis and ameliorates secondary lymphedema.

Although lymphedema is a common clinical condition, treatment for this disabling condition remains limited and largely ineffective. Recently, it has been reported that overexpression of VEGF-C correlates with increased lymphatic vessel growth (lymphangiogenesis). However, the effect of VEGF-C-induced lymphangiogenesis on lymphedema has yet to be demonstrated. Here we investigated the impact of local transfer of naked plasmid DNA encoding human VEGF-C (phVEGF-C) on two animal models of lymphedema: one in the rabbit ear and the other in the mouse tail. In a rabbit model, following local phVEGF-C gene transfer, VEGFR-3 expression was significantly increased. This gene transfer led to a decrease in thickness and volume of lymphedema, improvement of lymphatic function demonstrated by serial lymphoscintigraphy, and finally, attenuation of the fibrofatty changes of the skin, the final consequences of lymphedema. The favorable effect of phVEGF-C on lymphedema was reconfirmed in a mouse tail model. Immunohistochemical analysis using lymphatic-specific markers: VEGFR-3, lymphatic endothelial hyaluronan receptor-1, together with the proliferation marker Ki-67 Ab revealed that phVEGF-C transfection potently induced new lymphatic vessel growth. This study, we believe for the first time, documents that gene transfer of phVEGF-C resolves lymphedema through direct augmentation of lymphangiogenesis. This novel therapeutic strategy may merit clinical investigation in patients with lymphedema.

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 10(7) NA/CD31+ MNCs, 10(7) NA/CD31- MNCs, or PBS was performed with a NOGA mapping injection catheter to target ischemic myocardium. In a parallel study, 10(5) human CD34+ MNCs, 10(5) 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.

Transcutaneous ultrasound augments naked DNA transfection of skeletal muscle.

This study was designed to test the hypothesis that transcutaneous ultrasound (US) exposure may augment the transfection efficiency and biological outcome associated with nonviral DNA gene transfer. Hindlimb muscles of New Zealand White rabbits were transfected with the reporter plasmid pCMV-beta, with or without US exposure. Optimization studies employed US exposure at various frequencies, mechanical indices, duty cycles, durations of exposure, and exposure time points. Based on these results, we explored the effect of US exposure on nonviral gene transfer of vascular endothelial growth factor (VEGF, phVEGF165) to promote neovascularization of ischemic hindlimbs. Ultrasound at 1 MHz, 100 W/cm(2), 6% duty cycle, and 5 minutes exposure time, applied immediately following DNA injection, was found to be the most effective among the settings tested, increasing beta-galactosidase expression approximately 20 fold. Compared with US exposure alone, or phVEGF165 only, phVEGF165 + US exposure yielded a statistically significant improvement in revascularization, as determined by calf blood pressure ratio, angiographic score, intravascular Doppler blood flow, and capillary/myocyte ratio. These data demonstrate that ultrasound, when applied directly after intramuscular gene transfer, significantly increases transfection efficiency in vivo. The biological significance of this finding was confirmed by augmented limb perfusion in response to US exposure and naked VEGF DNA.

Plasma vascular endothelial growth factor (VEGF) levels after intramuscular and intramyocardial gene transfer of VEGF-1 plasmid DNA.

The purpose of this study was to document the kinetics of vascular endothelial growth factor (VEGF) protein release into the systemic circulation after phVEGF gene transfer for therapeutic angiogenesis. VEGF plasma levels were measured by ELISA in 64 patients undergoing gene transfer of plasmid DNA: intramuscular in 34 patients with peripheral artery disease, and intramyocardial in 30 patients with coronary disease. Baseline plasma VEGF was highly variable and not normally distributed. After intramuscular gene transfer, median plasma VEGF rose slightly, although significantly, by 7 days (38 to 41 pg/ml, p < 0.05), but was not different from baseline at 14, 21, or 28 days. After intramyocardial gene transfer, median plasma VEGF levels were significantly elevated compared with baseline on days 2, 3, and 7 (39, 38, and 45 pg/ml, respectively, each p < 0.05 vs. baseline value of 21 pg/ml). Day 7 plasma levels did not differ significantly as a function of phVEGF dose, or between intramyocardial and intramuscular injections (1.8 and 1.3 times baseline levels, respectively, p = 0.6), despite an almost 10-fold difference in mean phVEGF dose. Intramuscular and intramyocardial phVEGF injections result in significant, although modest, elevations of circulating gene product for <14 days, with no relationship to injected dose. While a statistically significant increase in circulating VEGF level can provide evidence of successful gene transfer for groups of patients, interpretation of results for individual subjects is complicated by wide variation in baseline VEGF and low circulating levels compared with baseline after gene transfer.