Recombinant PAI-1 therapy restores myoendothelial junctions and erectile function in PAI-1-deficient mice.

Myoendothelial junctions are specialised projections of cell : cell contact through the internal elastic lamina between endothelial cells and vascular smooth muscle cells. These junctions allow for endothelial cells and vascular smooth muscle cells to make direct membrane apposition and are involved in cell : cell communication. In this study, we evaluated for the presence of myoendothelial junctions in murine corporal tissue and used plasminogen activator inhibitor (PAI)-1-deficient mice, which lack myoendothelial junctions, to determine whether myoendothelial junctions affect erectile function. Transmission electron microscopy demonstrated the presence of myoendothelial junctions in the corporal tissue of wild-type mice and confirmed the decreased junction numbers in the tissue of PAI-1(-/-) mice. A potential role for myoendothelial junctions in tumescence was established; in that, PAI-1(-/-) mice demonstrated a significantly longer time to achieve maximal intracavernous pressure. Treatment of PAI-1(-/-) mice with recombinant PAI-1 restored the number of myoendothelial junctions in the corporal tissue and also induced a significant decrease in time to maximal corporal pressures. Myoendothelial junctions were similarly identified in the human corporal tissue. These results suggest a critical role for myoendothelial junctions in erectile pathophysiology and therapies aimed at restoring myoendothelial junction numbers in the corporal tissue may provide a novel therapy for erectile dysfunction.

Adeno-associated virus serotype 9 efficiently targets ischemic skeletal muscle following systemic delivery.

Targeting therapeutic gene expression to the skeletal muscle following intravenous (IV) administration is an attractive strategy for treating peripheral arterial disease (PAD), except that vector access to the ischemic limb could be a limiting factor. As adeno-associated virus serotype 9 (AAV-9) transduces skeletal muscle at high efficiency following systemic delivery, we employed AAV-9 vectors bearing luciferase or enhanced green fluorescent protein (eGFP) reporter genes to test the hypothesis that increased desialylation of cell-surface glycans secondary to hindlimb ischemia (HLI) might help offset the reduction in tissue perfusion that occurs in mouse models of PAD. The utility of the creatine kinase-based (CK6) promoter for restricting gene expression to the skeletal muscle was also examined by comparing it with the cytomegalovirus (CMV) promoter after systemic administration following surgically induced HLI. Despite reduced blood flow to the ischemic limbs, CK6 promoter-driven luciferase activities in the ischemic gastrocnemius (GA) muscles were ∼34-, ∼28- and ∼150-fold higher than in the fully perfused contralateral GA, heart and liver, respectively, 10 days after IV administration. Furthermore, luciferase activity from the CK6 promoter in the ischemic GA muscles was ∼twofold higher than with CMV, while in the liver CK6-driven activity was ∼42-fold lower than with CMV, demonstrating that the specificity of ischemic skeletal muscle transduction can be further improved with the muscle-specific promoters. Studies with Evans blue dye and fluorescently labeled lectins revealed that vascular permeability and desialylation of the cell-surface glycans were increased in the ischemic hindlimbs. Furthermore, AAV9/CK6/Luc vector genome copy numbers were ∼sixfold higher in the ischemic muscle compared with the non-ischemic muscle in the HLI model, whereas this trend was reversed when the same genome was packaged in the AAV-1 capsid (which binds sialylated, as opposed to desialylated glycans), further underscoring the importance of desialylation in the ischemic enhancement of transduction displayed by AAV-9. Taken together, these findings suggest two complementary mechanisms contributing to the preferential transduction of ischemic muscle by AAV-9: increased vascular permeability and desialylation. In conclusion, ischemic muscle is preferentially targeted following systemic administration of AAV-9 in a mouse model of HLI. Unmasking of the primary AAV-9 receptor as a result of ischemia may contribute importantly to this effect.

Inhibiting Anti-angiogenic VEGF165b Activates a Novel miR-17-20a-Calcipressin-3 Pathway that Revascularizes Ischemic Muscle in Peripheral Artery Disease.

Background: VEGF165a increases the expression of microRNA-17-92 cluster, promoting developmental, retinal, and tumor angiogenesis. We have previously shown that VEGF165b, an alternatively spliced VEGF-A isoform, inhibits the VEGFR-STAT3 pathway in ischemic endothelial cells (ECs) to decrease their angiogenic capacity. In ischemic macrophages (Møs), VEGF165b inhibits VEGFR1 to induce S100A8/A9 expression, which drives M1-like polarization. Our current study aims to determine whether VEGF165b inhibition promotes perfusion recovery by regulating the miR-17-92 cluster in preclinical PAD. Methods: Hind limb ischemia (HLI) induced by femoral artery ligation and resection was used as a preclinical PAD model. Hypoxia serum starvation (HSS) was used as an in vitro PAD model. VEGF165b was inhibited/neutralized by an isoform-specific VEGF165b antibody. Results: Systematic analysis of miR-17-92 cluster members (miR-17-18a-19a-19b-20a-92) in experimental-PAD models showed that VEGF165b-inhibition induces miRNA-17-20a (within miR-17-92 cluster) in HSS-ECs and HSS-bone marrow derived macrophages (BMDMs) vs. respective normal and/or isotype matched IgG controls to enhance perfusion-recovery. Consistent with the bioinformatics analysis that revealed RCAN3 as a common target of miR-17 and miR-20a, Argonaute-2 pull-down assays showed decreased miR-17-20a expression and higher RCAN3 expression in the RISC complex of HSS-ECs and HSS-BMDMs vs. the respective controls. Inhibiting miR-17-20a induced RCAN3 levels to decrease ischemic angiogenesis and promoted M1-like polarization to impair perfusion recovery. Finally, using STAT3 inhibitors, S100A8/A9 silencers and VEGFR1-deficient ECs and Møs, we show that VEGF165b inhibition activates the miR-17-20a-RCAN3 pathway independent of VEGFR1-STAT3 or VEGFR1-S100A8/A9 in ischemic ECs and ischemic Møs, respectively. Conclusion: Our data revealed a hereunto unrecognized therapeutic 'miR-17-20a-RCAN3' pathway in the ischemic vasculature that is VEGFR1-STAT3/S100A8/A9 independent and is activated only upon VEGF165b inhibition in PAD.

Exercise dose response in muscle.

Exercise increases peak VO2 partially through muscle adaptations. However, understanding muscle adaptations related to exercise dose is incomplete. This study investigated exercise training dose on capillaries per fiber and capillaries per area; and citrate synthase from vastus lateralis and related both to changes in peak VO2. This randomized trial compared 3 exercise doses: low amount-moderate intensity (n=40), low amount-high intensity (n=47), high amount-high intensity (n=41), and a control group (n=35). Both measures of capillary supply increased in all exercise groups (p<0.05). Low amount-high intensity and high amount-high intensity improved citrate synthase (p<0.05) and the low amount-moderate intensity citrate synthase approached significance (p=0.059). Muscle improvements were only related to improvements in peak VO2 in high amount-high intensity (citrate synthase, r=0.304; capillaries:fiber, r= - 0.318; p<0.05 and capillaries/mm2 r= - 0.310, p<0.05). These data suggest muscle adaptations occur following both low and high exercise doses, but are only related to improved peak VO2 following high amount-high intensity training.

Mitochondrial DNA structure and expression in specialized subtypes of mammalian striated muscle.

Mitochondrial DNA (mt DNA) in cells of vertebrate organisms can assume an unusual triplex DNA structure known as the displacement loop (D loop). This triplex DNA structure forms when a partially replicated heavy strand of mtDNA (7S mtDNA) remains annealed to the light strand, displacing the native heavy strand in this region. The D-loop region contains the promoters for both heavy- and light-strand transcription as well as the origin of heavy-strand replication. However, the distribution of triplex and duplex forms of mtDNA in relation to respiratory activity of mammalian tissues has not been systematically characterized, and the functional significance of the D-loop structure is unknown. In comparisons of specialized muscle subtypes within the same species and of the same muscle subtype in different species, the relative proportion of D-loop versus duplex forms of mtDNA in striated muscle tissues of several mammalian species demonstrated marked variation, ranging from 1% in glycolytic fast skeletal fibers of the rabbit to 65% in the mouse heart. There was a consistent and direct correlation between the ratio of triplex to duplex forms of mtDNA and the capacity of these tissues for oxidative metabolism. The proportion of D-loop forms likewise correlated directly with mtDNA copy number, mtRNA abundance, and the specific activity of the mtDNA (gamma) polymerase. The D-loop form of mtDNA does not appear to be transcribed at greater efficiency than the duplex form, since the ratio of mtDNA copy number to mtRNA was unrelated to the proportion of triplex mtDNA genomes. However, tissues with a preponderance of D-loop forms tended to express greater levels of cytochrome b mRNA relative to mitochondrial rRNA transcripts, suggesting that the triplex structure may be associated with variations in partial versus full-length transcription of the heavy strand.

Impaired collateral vessel development associated with reduced expression of vascular endothelial growth factor in ApoE-/- mice.

BACKGROUND: The impact of disordered lipid metabolism on collateral vessel development was studied in apolipoprotein (apo)E-/- mice with unilateral hindlimb ischemia. METHODS AND RESULTS: Hindlimb blood flow and capillary density were markedly reduced in apoE-/- mice versus C57 controls. This was associated with reduced expression of vascular endothelial growth factor (VEGF) in the ischemic limbs of apoE-/- mice. Cell-specific immunostaining localized VEGF protein expression to skeletal myocytes and infiltrating T cells in the ischemic limbs of C57 mice; in contrast, T-cell infiltrates in ischemic limbs of apoE-/- mice were severely reduced. The critical contribution of T cells to VEGF expression and collateral vessel growth was reinforced by the finding of accelerated limb necrosis in athymic nude mice with operatively induced hindlimb ischemia. Adenoviral VEGF gene transfer to apoE-/- mice resulted in marked augmentation of hindlimb blood flow and capillary density. CONCLUSIONS: These findings thus underscore the extent to which hyperlipidemia adversely affects native collateral development but does not preclude augmented collateral vessel growth in response to exogenous cytokines. Moreover, results obtained in the apoE-/- and athymic nude mice imply a critical role for infiltrating T cells as a source of VEGF in neovascularization of ischemic tissues.

Capillary density of skeletal muscle: a contributing mechanism for exercise intolerance in class II-III chronic heart failure independent of other peripheral alterations.

OBJECTIVES: The study was conducted to determine if the capillary density of skeletal muscle is a potential contributor to exercise intolerance in class II-III chronic heart failure (CHF). BACKGROUND: Previous studies suggest that abnormalities in skeletal muscle histology, contractile protein content and enzymology contribute to exercise intolerance in CHF. METHODS: The present study examined skeletal muscle biopsies from 22 male patients with CHF compared with 10 age-matched normal male control patients. Aerobic capacities, myosin heavy chain (MHC) isoforms, enzymes, and capillary density were measured. RESULTS: The patients with CHF demonstrated a reduced peak oxygen consumption when compared to controls (15.0+/-2.5 vs. 19.8+/-5.0 ml x kg(-1) x min(-1), p <0.05). Using cell-specific antibodies to directly assess vascular density, there was a reduction in capillary density in CHF measured as the number of endothelial cells/fiber (1.42+/-0.28 vs. 1.74+/-0.35, p = 0.02). In CHF, capillary density was inversely related to maximal oxygen consumption (r = 0.479, p = 0.02). The MHC IIx isoform was found to be higher in patients with CHF versus normal subjects (28.5+/-13.6 vs. 19.5+/-9.4, p <0.05). CONCLUSIONS: There was a significant reduction in microvascular density in patients with CHF compared with the control group, without major differences in other usual histologic and biochemical aerobic markers. The inverse relationship with peak oxygen consumption seen in the CHF group suggests that a reduction in microvascular density of skeletal muscle may precede other skeletal muscle alterations and play a critical role in the exercise intolerance characteristic of patients with CHF.

Transmyocardial laser revascularization limits in vivo adenoviral-mediated gene transfer in porcine myocardium.

OBJECTIVE: Transmyocardial laser revascularization (TMR) is emerging as a potential treatment option for patients with end-stage CAD, and adjuvant gene therapy may be helpful in further improving the results of the procedure. However, the effects of TMR on gene transfer are unknown. METHODS: Swine underwent left thoracotomy. TMR was performed to create five channels at 2-cm intervals in the anterolateral free wall of the left ventricle (LV) followed by injection of 1 x 10(9) plaque-forming units (pfu) of a replication-deficient adenovirus vector carrying the reporter gene beta-galactosidase (Ad.Pac beta-gal). An additional five direct injections of 1 x 10(9) pfu Ad.Pac beta-gal were made at 2-cm intervals in the posterolateral LV of each heart. Control animals underwent TMR alone/vehicle alone (n = 3) or empty virus alone/no treatment (n = 3) of the anterolateral/posterolateral LV, respectively. RESULTS: ELISA revealed significantly greater transgene expression in the direct Ad.Pac beta-gal injection versus TMR plus Ad.Pac beta-gal inject regions at both 3 (n = 6) (273.0 +/- 58.5 vs. 133.4 + 28.1 pg beta-gal/g protein, P = 0.02) and 7 days (n = 6) (180.0 + 59.9 vs. 56.7 + 18.1 pg beta-gal/g protein, P = 0.02) postoperatively. At 14 days postoperatively (n = 2), no transgene expression was detected in either region. No transgene expression was detected in any of the control regions at 3 days postoperatively. CD-18 staining revealed significantly greater inflammation in the TMR plus Ad.Pac beta-gal and TMR alone regions as compared to Ad.Pac beta-gal or vehicle (P < 0.001). CONCLUSIONS: Adenoviral-mediated gene transfer in conjunction with TMR is possible, although TMR appears to limit the degree of transgene expression attained as compared to direct intramyocardial injection alone, likely due to the greater immune response observed with the former. These findings may have important implications for therapeutic strategies aimed at combining TMR with gene therapy for CAD.

Efficient adenoviral gene transfer to early venous bypass grafts: comparison with native vessels.

OBJECTIVES: Gene therapy may provide new approaches to reduce vein graft failure following coronary or peripheral bypass surgery. The aim of this study was to investigate the relative efficacy of intraoperative adenoviral gene transfer to vein grafts, comparing transgene expression in vein grafts with that in matched native vessels in the same animal. In addition, we assessed the impact of bypass grafting on the cellular targets of gene transfer. METHODS: New Zealand White rabbits underwent interposition bypass grafting of the carotid artery, using the ipsilateral external jugular vein, which was infected with an adenovirus expressing beta-galactosidase immediately prior to bypass grafting (n = 16). The contralateral native jugular vein (n = 16) and carotid artery (n = 8) were infected concurrently with the same adenoviral preparation. After 3, 7 or 14 days, beta-galactosidase protein expression was quantified by ELISA, and specific cell types expressing beta-galactosidase were identified by X-Gal staining and by immunohistochemistry. RESULTS: After 3 days, endothelial cells were efficiently transduced in all vessels; medial smooth muscle cells were transduced infrequently. In contrast to jugular veins after gene transfer, endothelium in vein grafts showed expression of VCAM-1 and ICAM-1, and intense inflammation with CD18+ leukocytes. Transgene expression in vein grafts at day 3 was maintained at levels approximately 50% of that in ungrafted jugular veins, but continued to decrease through day 7. CONCLUSIONS: Although vascular injury in early venous bypass grafts reduces gene transfer efficacy, significant transgene expression is maintained for at least 7 days. These findings have important implications for intraoperative gene transfer strategies in vein grafts.

Collagen subtypes III and IV expression in human vein graft atherosclerosis.

This study examined the expression of collagen subtypes III and IV in a series of freshly excised human venous coronary artery bypass grafts. The results of this study demonstrate that these collagen subtypes are differentially expressed in vein graft atherosclerosis.

Design of the therapeutic angiogenesis with recombinant fibroblast growth factor-2 for intermittent claudication (TRAFFIC) trial.

The Therapeutic Angiogenesis With Recombinant Fibroblast Growth Factor-2 for Intermittent Claudication (TRAFFIC) is a large, randomized, placebo-controlled, regimen-finding trial of intra-arterial recombinant fibroblast growth factor-2 in patients with intermittent claudication. This report describes the major design considerations and end points in TRAFFIC.

Differences in skeletal muscle between men and women with chronic heart failure.

Men with chronic heart failure (CHF) have alterations in their skeletal muscle that are partially responsible for a decreased exercise tolerance. The purpose of this study was to investigate whether skeletal muscle alterations in women with CHF are similar to those observed in men and if these alterations are related to exercise intolerance. Twenty-five men and thirteen women with CHF performed a maximal exercise test for evaluation of peak oxygen consumption (VO(2)) and resting left ventricular ejection fraction, after which a biopsy of the vastus lateralis was performed. Twenty-one normal subjects (11 women, 10 men) were also studied. The relationship between muscle markers and peak VO(2) was consistent for CHF men and women. When controlling for gender, analysis showed that oxidative enzymes and capillary density are the best predictors of peak VO(2.) These results indicate that aerobically matched CHF men and women have no differences in skeletal muscle biochemistry and histology. However, when CHF groups were separated by peak exercise capacity of 4.5 metabolic equivalents (METs), CHF men with peak VO(2) >4.5 METs had increased citrate synthase and 3-hydroxyacyl-CoA dehydrogenase compared with CHF men with peak VO(2) <4.5 METs. CHF men with a lower peak VO(2) had increased capillary density compared with men with higher peak VO(2). These observations were not reproduced in CHF women. This suggests that differences may exist in how skeletal muscle adapts to decreasing peak VO(2) in patients with CHF.

High-flow gas insufflation to facilitate MIDCABG: effects on coronary endothelium.

BACKGROUND: During less invasive coronary bypass operations on the beating heart, as well as conventional operations using continuous warm cardioplegia, a precise anastomosis is facilitated by a bloodless field. To maintain a clear field, many surgeons use high-flow gas insufflation. However, the potentially damaging effects of gas insufflation on coronary endothelium have not been elucidated. METHODS: Seven pigs underwent median sternotomy. Between two coronary occluders, an arteriotomy in the mid left anterior descending coronary artery (LAD) was performed. In the experimental group (n = 5), the operative field was kept clear by exposing the arteriotomy to a catheter-directed stream of carbon dioxide at 15 L/min. In the control group (n = 2), the arteriotomy was left open to room air. After 20 minutes, the segments of LAD exposed to carbon dioxide or room air, and the unexposed proximal LAD and right coronary artery, were processed, sectioned, and stained together. A murine anti-human tie-2 monoclonal antibody was used to identify endothelium. RESULTS: All unexposed LAD and right coronary artery segments and all LAD segments exposed only to room air demonstrated normal, contiguous staining of endothelium with the murine anti-human tie-2 monoclonal antibody. In contrast, all LAD segments exposed to high-flow carbon dioxide gas insufflation demonstrated near-complete loss of endothelium. CONCLUSIONS: These data demonstrate that high-flow carbon dioxide gas insufflation denudes the coronary artery of its endothelium. This exposes blood elements to the subendothelium and promotes clotting, and endothelial loss may promote smooth muscle cell migration and proliferation. These events set the stage for early and late graft failure.

Induction of angiogenesis after TMR: a comparison of holmium: YAG, CO2, and excimer lasers.

BACKGROUND: Transmyocardial laser revascularization (TMR) is an emerging treatment for end-stage coronary artery disease. A variety of lasers are currently available to perform the procedure, although their relative efficacy is unknown. The purpose of this study was to compare changes in myocardial blood flow and function 6 months after TMR with holmium:yttrium-aluminum-garnet (holmium:YAG), carbon dioxide (CO2), and xenon chloride excimer lasers in a model of chronic ischemia. METHODS: Miniswine underwent subtotal (90%) left circumflex coronary stenosis. Baseline positron emission tomography and dobutamine stress echocardiography were performed to document hibernating myocardium in the left circumflex coronary artery distribution. Animals were then randomized to sham redo-thoracotomy (n = 5) or TMR using a holmium:YAG (n = 5), CO2 (n = 5) or excimer (n = 5) laser. Six months postoperatively, the positron emission tomography and dobutamine stress echocardiography studies were repeated and the animals sacrificed. RESULTS: In animals undergoing TMR with holmium: YAG and CO2 lasers, a significant improvement in myocardial blood flow to the lased left circumflex regions was seen. No significant change in myocardial blood flow was seen in sham- or excimer-lased animals. There was a significant improvement in regional stress function of the lased segments 6 months postoperatively in animals undergoing holmium:YAG and CO2 laser TMR that was consistent with a reduction in ischemia. There was no change in wall motion in sham- or excimer-lased animals. Significantly greater neovascularization was observed in the holmium:YAG and CO2 lased regions than with either the sham procedure or excimer TMR. CONCLUSIONS: Transmyocardial laser revascularization with either holmium:YAG or CO2 laser improves myocardial blood flow and contractile reserve in lased regions 6 months postoperatively. These changes were not seen following excimer TMR or sham thoracotomy, suggesting that differences in laser energy or wavelength or both may be important in the induction of angiogenesis.

Improved perfusion and contractile reserve after transmyocardial laser revascularization in a model of hibernating myocardium.

BACKGROUND: Transmyocardial laser revascularization (TMR) has been demonstrated effective for relieving angina, although prior studies have yielded inconsistent results regarding postoperative myocardial perfusion and function. This study evaluated long-term changes in myocardial perfusion and contractile reserve after TMR in a model of hibernating myocardium. METHODS: Miniswine had subtotal left circumflex coronary artery occlusion to reduce resting blood flow to 10% of baseline. After 2 weeks in the low-flow state, positron emission tomography and dobutamine stress echocardiography were performed to document ischemic, viable (hibernating) myocardium in the left circumflex distribution. Animals then had sham redo thoracotomy (n = 4) or TMR (n = 6). Six months later the positron emission tomography and dobutamine stress echocardiography studies were repeated. RESULTS: Myocardial blood flow in the left circumflex distribution as measured by positron emission tomography was significantly reduced in all animals after 2 weeks in the low-flow state. In animals that had TMR, there was significant improvement in myocardial blood flow to the lased regions 6 months postoperatively. No significant change in myocardial blood flow was seen in sham animals at 6 months. Dobutamine stress echocardiography after 2 weeks of low-flow demonstrated severe hypocontractility at rest in the left circumflex region of all animals, with a biphasic response to dobutamine consistent with hibernating myocardium. In animals that had TMR, there was a trend toward improved resting function and significantly improved regional stress function in the lased segments 6 months postoperatively, consistent with a reduction in ischemia. Global left ventricular wall motion at peak stress improved significantly as well. There was no change in wall motion 6 months postoperatively in sham-operated animals. CONCLUSIONS: This study found improvements in myocardial perfusion and regional and global contractile reserve 6 months after TMR in a porcine model of hibernating myocardium. This improved perfusion and function likely accounts for the clinical benefits of the procedure.

Neovascularization after transmyocardial laser revascularization in a model of chronic ischemia.

BACKGROUND: The mechanism of clinical improvement after transmyocardial laser revascularization (TMR) is unknown. One hypothesis holds that TMR causes increased myocardial perfusion through neovascularization. This study sought to determine whether angiogenesis occurs after TMR in a porcine model of chronic myocardial ischemia. METHODS: Six miniature pigs underwent subtotal left circumflex coronary artery occlusion to reduce resting blood flow to 10% of baseline. After 2 weeks in the low-flow state, dobutamine stress echocardiography and positron emission tomography were performed to document ischemic, viable myocardium. The animals then underwent TMR and were sacrificed 6 months later for histologic and immunohistochemical analysis. RESULTS: Histologic analysis of the lased left circumflex region demonstrated many hypocellular areas filled with connective tissue representing remnant TMR channels. Histochemical staining demonstrated a highly disorganized pattern of neovascularization consistent with angiogenesis located predominantly at the periphery of the channels. Immunohistochemical analysis confirmed the presence of endothelial cells within neovessels. Vascular density analysis revealed a mean of 29.2+/-3.6 neovessels per high-power field in lased ischemic myocardium versus 4.0+/-0.3 (p<0.001) in nonlased ischemic myocardium. CONCLUSIONS: This study provides evidence that neovascularization is present long term in regions of ischemic, viable myocardium after TMR. Angiogenesis may represent the mechanism of clinical improvement after TMR.

Intracavernosal injections of vascular endothelial growth factor protects endothelial dependent corpora cavernosal smooth muscle relaxation in the hypercholesterolemic rabbit: a preliminary study.

Atherosclerosis is a major risk factor for erectile dysfunction, and loss of endothelium-dependent vasodilation appears early in the development of this disorder. Nitric oxide (NO) appears to be the principle mediator of erectile function and is generated in part by the sinusoidal endothelium. Vascular endothelial growth factor (VEGF) is an angiogenic growth factor and an endothelial cell-specific mitogen and the actions of VEGF are coupled to NO. In this preliminary study, we investigated whether VEGF could be used to protect endothelial dependent cavernosal relaxation from the atherosclerotic injury induced by a hypercholesterolemic diet.Two groups of New Zealand white adult male rabbits received a 1% cholesterol diet for four weeks, and two groups consumed normal rabbit chow. Half of the rabbits consuming the 1% cholesterol diet received weekly penile injections of 0.3 mg VEGF (n=8), and half injections of normal saline (n=8). Rabbits fed normal chow followed a similar protocol, half received weekly penile injections of 0.3 mg VEGF (n=6) and half were given weekly penile injections of normal saline (n=6). Isometric tension studies (with norepinephrine, acetylcholine, sodium nitroprusside and histamine) were performed on isolated strips of corpora cavernosa. The degree of corporal smooth muscle relaxation in response to ACH and SNP administration was recorded and compared. Significant elevation in serum total cholesterol levels occurred in rabbits receiving 4 weeks of the 1% cholesterol diet (727+/-75.6 mg/dl vs 38.7+/-5.53 mg/dl) P<0.01. There were no significant differences in cavernosal contraction in any group, while cavernosal smooth muscle from rabbits on normal chow retained the ability to relax in response to ACH and SNP in tissue bath. The hypercholesterolemic rabbits receiving VEGF had a significantly higher maximal per-cent relaxation to ACH (111+/-28.9) compared to the hypercholesterolemic rabbits that received NS (77+/-23.1, P<0.001). This difference in percent maximal relaxation to SNP was also present for hypercholesterolemic/VEGF rabbits (129.4+/-24) versus the hypercholesterolemic/NS rabbits (115.0+/-18, P=0.033). In conclusion, intracavernosal injections of VEGF appear to protect corporal endothelium from hypercholesterolemia induced injury, thus preserving endothelial dependent corporal smooth muscle relaxation in hypercholesterolemic rabbit.

Alterations in collagen subtype III and IV protein in experimental venous bypass grafting.

OBJECTIVE: To examine changes in collagen III and IV protein during the development of intimal hyperplasia in experimental vein grafts. METHODS: Sixteen New Zealand White rabbits underwent reversed, jugular vein, interposition grafting of the carotid artery. Vessels were harvested 3, 7 or 28 days after operation and subjected to immunohistochemical examination and gelatinase assays. RESULTS: In control vein, collagen IV was expressed around adventitial blood vessels and throughout the endothelium. Compared with its presence in control veins, collagen IV protein was decreased in the endothelium in all 3-day vein grafts and undetectable in the endothelium and intima in 7-day vein grafts, but was present in the endothelium and intimal hyperplasia in 28-day vein grafts. In contrast, collagen III was absent from the endothelium of control vein and 3-day vein grafts, was present at low levels in the intima of 7-day vein grafts, but was absent from the endothelium and intimal hyperplasia in 28-day vein grafts. In 3-day vein grafts, areas of collagen IV loss colocalized to areas of leukocyte infiltration. Protein extracts from 3-day vein grafts contained a 72 kDa gelatinase. CONCLUSIONS: The presence and alterations of collagen protein in veins and vein grafts are subtype specific. Collagen III does not appear to be a normal component of intimal hyperplasia in vein grafts. The decrease in collagen IV protein in the endothelium of veins may be a component of the endothelial changes that follow bypass grafting, mediated by leukocytes, the induction of gelatinase activity, or both.

Coronary angioscopy. Clinical applications.

Percutaneous coronary angioscopy is an invaluable alternative imaging modality that can not be used routinely in the interventional cardiac catheterization laboratory. The major value of angioscopy lies in its ability to determine safely, quickly, and accurately intraluminal morphologies before and after coronary interventions. The results from angioscopy can then be used to target more specific treatment strategies, given the vast array of interventional treatment strategies available. Percutaneous coronary angioscopy, however, requires meticulous attention for the proper preparation and use of the angioscope to minimize the potential risks associated with the imaging procedure.

Transluminal extraction coronary atherectomy.

The TEC is a forward-cutting atherectomy catheter that has the unique potential to excise and aspirate atheroma, especially intraluminal thrombus. This device has been under clinical investigation for more than 6 years and received final marketing approval by the FDA for the treatment of lesions in the coronary vasculature in 1993. In the US TEC Multicenter Registry, the overall clinical and lesion successes were favorable. The success rates were similar for both native coronary vessels and saphenous vein bypass grafts. Importantly, the procedural success rates were maintained even in the presence of several unfavorable angiographic features, such as ostial location, intraluminal thrombus, and total occlusion. Further insights into the mechanisms of action of the TEC were made from studies using percutaneous angioscopy and intravascular ultrasound. Angioscopy clearly demonstrated that the TEC was indeed able to remove intraluminal thrombus, especially loosely adherent red thrombus, in a population of patients with unstable coronary syndromes. However, the TEC frequently leaves behind multiple intimal disruptions which not only create a possible nidus for restenosis but also explain the frequent hazy angiographic appearance of the vessel after TEC atherectomy. As is true for all new interventional devices, the specific niche for the TEC in interventional cardiology will best be determined in randomized clinical trials. There are several areas in which the TEC appears promising. First, this device may have a role in the management of patients with diffusely diseased saphenous vein grafts. Second, the TEC may be an effective primary therapy in lesions with intraluminal thrombus. Treatment with the TEC may then be followed by an adjunctive therapy to maximize the final vessel diameters. Finally, the TEC may be valuable in the treatment of lesions in patients with high-risk unstable coronary syndromes.