Search

Background: Patients with severe myocardial ischemia who are not candidates for percutaneous or surgical revascularization have few therapeutic options. Therapeutic angiogenesis in animal models with use of recombinant human vascular endothelial growth factor (rhVEGF) has resulted in successful revascularization of ischemic myocardium. This was a dose escalation trial designed to determine the safety and tolerability of intracoronary rhVEGF infusions., Methods and Results: Patients were eligible if they had stable exertional angina, a significant reversible perfusion defect by stress myocardial perfusion study, and coronary anatomy that was suboptimal for percutaneous coronary intervention or coronary artery bypass grafting. rhVEGF was administered to a total of 15 patients by 2 sequential (eg, right and left) intracoronary infusions, each for 10 minutes, at rates of 0.005 (n = 4), 0.017 (n = 4), 0.050 (n = 4), and 0.167 mg/kg/min (n = 3). Pharmacokinetic sampling and hemodynamic monitoring were performed for 24 hours. Radionuclide myocardial perfusion imaging was performed before treatment and at 30 and 60 days after treatment. Follow-up angiograms were performed on selected patients at 60 days. The maximally tolerated intracardiac dose of rhVEGF was 0.050 mg/kg/min. Minimal hemodynamic changes were seen at 0.0050 mg/kg/min (2% +/- 7% [SD] mean decrease in systolic blood pressure from baseline to nadir systolic blood pressure), whereas at 0.167 mg/kg/min there was a 28% +/- 7% mean decrease from baseline to nadir (136 to 95 mm Hg systolic). Myocardial perfusion imaging was improved in 7 of 14 patients at 60 days. All 7 patients with follow-up angiograms had improvements in the collateral density score., Conclusion: rhVEGF appears well tolerated by coronary infusion at rates up to 0.050 mg/kg/min. This study provides the basis for future clinical trials to assess the clinical benefit of therapeutic angiogenesis with rhVEGF.

The role of the cardiac myocyte as a mediator of paracrine signaling in the heart has remained unclear. To address this issue, we generated mice with cardiac myocyte-specific deletion of the vascular endothelial growth factor gene, thereby producing a cardiomyocyte-specific knockout of a secreted factor. The hearts of these mice had fewer coronary microvessels, thinned ventricular walls, depressed basal contractile function, induction of hypoxia-responsive genes involved in energy metabolism, and an abnormal response to beta-adrenergic stimulation. These findings establish the critical importance of cardiac myocyte-derived vascular endothelial growth factor in cardiac morphogenesis and determination of heart function. Further, they establish an adult murine model of hypovascular nonnecrotic cardiac contractile dysfunction.

The initiating factors in angiogenesis during development and disease are often microenvironmental changes, which induce signaling to the vasculature from affected tissues. Among these, lowered oxygen pressure, hypoxia, is one of the most potent inducers/initiators of an angiogenic response. Significant evidence indicates that hypoxia acts as a morphogen during vascularization - inducing and shaping the recruitment and formation of new vascular beds through critical transcriptional control pathways. Recent advances indicate that extensive interactions occur between developing blood vessels, the tissues that they vascularize, and the interstitial environment to control and shape the establishment of new capillary beds. Identification of the processes that control the hypoxic response intracellularly has allowed an increasingly sophisticated understanding of angiogenesis as a process that is very closely tied to the microenvironment that it occurs in. Further understanding of these processes may present powerful therapeutic opportunities for disease intervention.

The rapid development of angiogenic growth factor therapy for patients with advanced ischemic heart disease over the last 5 years offers hope of a new treatment strategy based on generation of new blood supply in the diseased heart. However, as the field of therapeutic coronary angiogenesis is maturing from basic and preclinical investigations to clinical trials, many new and presently unresolved issues are coming into focus. These include in-depth understanding of the biology of angiogenesis, selection of appropriate patient populations for clinical trials, choice of therapeutic end points and means of their assessment, choice of therapeutic strategy (gene versus protein delivery), route of administration, and the side effect profile. The present article presents a summary statement of a panel of experts actively working in the field, convened by the Angiogenesis Foundation and the Angiogenesis Research Center during the 72nd meeting of the American Heart Association to define and achieve a consensus on the challenges facing development of therapeutic angiogenesis for coronary disease.

We describe two patients with spontaneous coronary artery dissection (SCAD) while on immunosuppressive therapy following renal transplantation. The role of cyclosporine A as a potential etiologic factor in spontaneous coronary artery dissection is discussed. A review of the recent literature on spontaneous coronary artery dissection highlights the changing clinical presentation and management of these patients.

The force-frequency relationship (FFR) describes the frequency-dependent potentiation of cardiac contractility. The interaction of the sarcoplasmic reticulum Ca2+-adenosinetriphosphatase (SERCA2) with its inhibitory protein phospholamban (PLB) might be involved in the control of the FFR. The FFR was analyzed in two systems in which the PLB-to-SERCA2 ratio was modulated. Adult rabbit cardiac myocytes were transduced with adenovirus encoding for SERCA2, PLB, and beta-galactosidase (control). After 3 days, the relative PLB/SERCA2 values were significantly different between groups (SERCA2, 0.5; control, 1.0; PLB, 4.5). SERCA2 overexpression shortened relaxation by 23% relative to control, whereas PLB prolonged relaxation by 39% and reduced contractility by 47% (0.1 Hz). When the stimulation frequency was increased to 1.5 Hz, myocyte contractility was increased by 30% in control myocytes. PLB-overexpressing myocytes showed an augmented positive FFR (+78%), whereas SERCA2-transduced myocytes displayed a negative FFR (-15%). A more negative FFR was also found in papillary muscles from SERCA2 transgenic mice. These findings demonstrate that the ratio of phospholamban to SERCA2 is an important component in the control of the FFR.

Background: We have previously reported that fosB mRNA is induced by hypertrophic stimuli (thrombin, angiotensin II) but not proliferative stimuli (platelet-derived growth factor, basic fibroblast growth factor) in pulmonary arterial smooth muscle cells (PASMCs) (J Biol Chem. 1994;9:6399-6404). Our aim in the present study was to investigate the potential role of FosB in PASMC hypertrophy., Methods and Results: Adenoviruses carrying sense or antisense fosB RNA expression cassettes were used to infect cultured PASMCs with the aim of increasing or inhibiting fosB expression, respectively. We examined whether fosB expression modification affected the growth of quiescent PASMCs, thrombin-induced hypertrophy, or platelet-derived growth factor-induced proliferation. PASMC growth was assessed by daily cell number count, determination of [3H]leucine incorporation, and quantification of total cellular protein. Neither an increase nor a decrease in FosB protein expression caused a significant change in the growth of quiescent PASMCs over a period of 96 hours, indicating that FosB alone is not sufficient to induce hypertrophy. Modification of FosB levels did not affect platelet-derived growth factor-induced PASMC proliferation. An increase in FosB expression did not augment thrombin-induced hypertrophy; however, inhibition of FosB expression resulted in a diminution of thrombin-induced hypertrophy by 58+/-6% (P<0.005)., Conclusions: These results suggest that FosB is necessary but not sufficient for thrombin-induced hypertrophy in cultured PASMCs.

Background: Decreased expression of the sarcoplasmic reticulum (SR) Ca2+-ATPase of the cardiac myocyte (SERCA2) and abnormal Ca2+ regulation have been independently linked to human heart failure. This study was designed to determine whether expression of a SERCA2 transgene could reconstitute depressed cardiac myocyte SERCA2 levels, augment SR Ca2+ uptake, and shorten prolonged excitation-contraction (EC)-associated Ca2+ transients in neonatal rat cardiac myocytes (NM)., Methods and Results: Cultured NM were treated with phorbol-12-myristate-13-acetate (PMA), a compound that decreases endogenous SERCA2 expression and results in prolongation of EC-associated Ca2+ transients. PMA-treated NM had a 75% reduction in SERCA2 mRNA and a 40% reduction in SERCA2 protein levels. SERCA2 adenovirus infection increased SERCA2 mRNA expression to 2.5 times control and reconstituted SERCA2 protein levels in PMA-treated cells. This reconstitution was associated with a 32.4% reduction in the time for decline of the Indo-1 Ca2+ transient to half-maximum levels (t(1/2) [Ca2+]i) (P<.05). A 34.5% augmentation of oxalate-facilitated SR Ca2+ uptake was also documented in SERCA2 adenovirus-infected cells (P<.05)., Conclusions: Adenovirus-mediated expression of a SERCA2 transgene can reconstitute depressed endogenous SERCA2 levels, shorten prolonged Ca2+ transients, and augment SR Ca2+ uptake. It is conceivable that such an approach might be used in vivo to normalize altered Ca2+ regulation in human heart failure.

We have recently shown that the overexpression of a heat shock protein 70 (hsp 70) in a rat myogenic cell line confers protection against simulated ischemia. We also developed and demonstrated that overexpression of this protein, in the hearts of transgenic mice, protects against ischemia/reperfusion injury. We have now inserted the hsp70 gene in an adenoviral vector and show that we are able to transfer and achieve overexpression of this protein in neonatal cardiomyocytes and in the rat myogenic cell line H9c2. We find that cells infected with the adenoviral-hsp70i construct are rendered tolerant to simulated ischemia as compared to cells infected with a control recombinant adenoviral construct. In conclusion, our results demonstrate the feasibility of using adenoviral vectors to overexpress the hsp70 in myogenic cells, specially in cardiomyocytes, and the efficiency of this approach for providing protection against myocardial ischemia.

Modification of gene expression in pulmonary arterial smooth muscle cells (PASMCs) could be a valuable tool for investigating the role of specific gene products in normal and pathological PASMC growth, and a novel potential therapy for pulmonary vascular diseases. To examine the direct role of fosB protein in PASMC growth, adenovirus (Ad) vectors were used to transfer sense or antisense full-length fosB cDNAs to cultured PASMCs to modify fosB expression, and investigate the effects of this modification on PASMC growth. The full-length fosB cDNA under the control of the cytomegalovirus (CMV) early gene promoter was constructed into an E1 region-deleted, replication-deficient human type 5 Ad vector in either sense or antisense orientation. Forty-eight hours after infection with the sense construct (Ad.S. fosB) at 3 plaque-forming units per cell (pfu/cell). PASMCs expressed abundant fosB mRNA and fosB protein. FosB protein was detected by immunohistochemistry in approximately 95% of the infected cells. PASMCs infected with Ad.S.fosB at ratios of 0.1, 0.2, 0.5, 1.3, and 10 pfu/cell showed a dose-dependent increase in fosB mRNA expression, with half-maximal and maximal expression at 1 and 10 pfu/cell, respectively. The increase in fosB mRNA expression was detected as early as 8 h and persisted for 25 days after infection. Forty-eight hours after infection with the antisense construct (Ad.A.fosB) at 3 pfu/cell, very low levels of fosB mRNA were detected by Northern blotting, in which the double-stranded fosB cDNA was labeled and used as a hybridization probe. FosB protein was undetectable by Western blotting or immunocytochemical analyses in the Ad.A.fosB infected cells. Cytopathical effects were observed when PASMCs were infected with either Ad.S.fosB or Ad.A.fosB at ratios of 10 pfu/cell or higher. Infection of serum-deprived PASMCs with Ad.S.fosB or Ad.A.fosB alone at 3 pfu/cell did not affect cellular growth. These results show that adenoviral vectors containing sense or antisense fosB cDNA expression units can be used to effectively modify fosB gene expression. Although changes in fos-B gene expression did not affect cellular growth, this model system offers a very effective method for elucidating the biological roles of other gene products and studying the pathways of PASMC gene regulation and signal transduction.

Increased coronary blood vessel development could potentially benefit patients with ischemic heart disease. In a model of stress-induced myocardial ischemia, intracoronary injection of a recombinant adenovirus expressing human fibroblast growth factor-5 (FGF-5) resulted in messenger RNA and protein expression of the transferred gene. Two weeks after gene transfer, regional abnormalities in stress-induced function and blood flow were improved, effects that persisted for 12 weeks. Improved blood flow and function were associated with evidence of angiogenesis. This report documents, for the first time, successful amelioration of abnormalities in myocardial blood flow and function following in vivo gene transfer.

Transforming growth factor-beta 1 (TGF-beta 1) is known to regulate cardiac cell function and its overexpression in the heart is thought to contribute to the development of cardiac hypertrophy and fibrosis. We wished to develop a high efficiency gene transfer method that could be used both in vitro and in vivo and result in the overexpression of TGF-beta 1. For this purpose, we constructed a replication-deficient human adenovirus 5 vector encoding for human TGF-beta 1 and used for control purposes an adenovirus lacZ vector. The adenovirus 5 construct was capable of infecting neonatal rat cardiac myocytes, fibroblasts and VSMCs. Of the three cell types, cardiac myocytes appear more susceptible to infection by the adenovirus 5 construct as assessed through beta-galactosidase staining. Infection of cardiac fibroblasts, myocytes and VSMCs with the hTGF-beta 1 adenovirus leads to the expression of hTGF-beta 1 mRNA and enhanced levels of bioactive and total TGF-beta 1 protein. Infection with hTGF-beta 1 adenovirus also results in enhanced levels of collagen type III gene expression in VSMCs and fibroblasts whereas in cardiac myocytes it leads to increased levels for sarcomeric and beta-actin. Thus, this adenoviral vector might be used for the exploration of in vivo effects of altered levels of cardiac TGF-beta 1.