Reed Hickey

Stromal Cell–Derived Factor-1α Plays a Critical Role in Stem Cell Recruitment to the Heart After Myocardial Infarction but Is Not Sufficient to Induce Homing in the Absence of Injury

Background—After myocardial infarction (MI), bone marrow–derived cells (BMDCs) are found within the myocardium. The mechanisms determining BMDC recruitment to the heart remain unclear. We investigated the role of stromal cell–derived factor-1α (SDF-1) in this process. Methods and Results—MI produced in mice by coronary ligation induced SDF-1 mRNA and protein expression in the infarct and border zone and decreased serum SDF-1 levels. By quantitative polymerase chain reaction, 48 hours after intravenous infusion of donor-lineage BMDCs, there were 80.5±15.6% more BDMCs in infarcted hearts compared with sham-operated controls (P<0.01). Administration of AMD3100, which specifically blocks binding of SDF-1 to its endogenous receptor CXCR4, diminished BMDC recruitment after MI by 64.2±5.5% (P<0.05), strongly suggesting a requirement for SDF-1 in BMDC recruitment to the infarcted heart. Forced expression of SDF-1 in the heart by adenoviral gene delivery 48 hours after MI doubled BMDC recruitment over MI alone (P<0.001) but did not enhance recruitment in the absence of MI, suggesting that SDF-1 can augment, but is not singularly sufficient for, BDMC recruitment to the heart. Gene expression analysis after MI revealed increased levels of several genes in addition to SDF-1, including those for vascular endothelial growth factor, matrix metalloproteinase-9, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, which might act in concert with SDF-1 to recruit BMDCs to the injured heart. Conclusion—SDF-1/CXCR4 interactions play a crucial role in the recruitment of BMDCs to the heart after MI and can further increase homing in the presence, but not in the absence, of injury.

A cardiac myocyte vascular endothelial growth factor paracrine pathway is required to maintain cardiac function

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 β-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.

Isoforms of Vascular Endothelial Growth Factor Act in a Coordinate Fashion To Recruit and Expand Tumor Vasculature

ABSTRACT Vascular endothelial growth factor (VEGF) is an essential regulator of vascularization. It is expressed as several splice variants; the major forms contain 120 amino acids, 164 amino acids, and 188 amino acids. We utilized transformed cells nullizygous for VEGF to specifically express each of these isoforms in isolation, in order to determine the role of each in tumorigenic neo-vascularization. We found that only the intermediate isoform, VEGF164, could fully rescue tumor growth; VEGF120 partially rescued tumor growth, and VEGF188 failed completely to rescue tumor expansion. Surprisingly, the vascular density of VEGF188 isoform-expressing tumors is significantly greater than that of wild-type VEGF cells and the other isoform-specific tumors. The failure of the hypervascular VEGF188-expressing tumors to grow may be due to inadequate perfusion of the massive number of microvessels in these tumors; three-dimensional imaging of the tumorigenic vasculature indicated little or no recruitment of the peripheral vasculature. This demonstrates that the VEGF isoforms perform unique functions which together enable tumorigenic vascularization.

Induction of angiogenesis in a mouse model using engineered transcription factors

The relationship between the structure of zinc-finger protein (ZFP) transcription factors and DNA sequence binding specificity has been extensively studied. Advances in this field have made it possible to design ZFPs de novo that will bind to specific targeted DNA sequences. It has been proposed that such designed ZFPs may eventually be useful in gene therapy. A principal advantage of this approach is that activation of an endogenous gene ensures expression of the natural array of splice variants. Preliminary studies in tissue culture have validated the feasibility of this approach. The studies reported here were intended to test whether engineered transcription factors are effective in a whole-organism model. ZFPs were designed to regulate the endogenous gene encoding vascular endothelial growth factor-A (Vegfa). Expression of these new ZFPs in vivo led to induced expression of the protein VEGF-A, stimulation of angiogenesis and acceleration of experimental wound healing. In addition, the neovasculature resulting from ZFP-induced expression of Vegfa was not hyperpermeable as was that produced by expression of murine Vegfa164 cDNA. These data establish, for the first time, that specifically designed transcription factors can regulate an endogenous gene in vivo and evoke a potentially therapeutic biophysiologic effect.

Cardiac myocyte-specific HIF-1α deletion alters vascularization, energy availability, calcium flux, and contractility in the normoxic heart

At a resting pulse rate the heart consumes almost twice‐as much oxygen per gram tissue as the brain and more than 43 times more than resting skeletal muscle (1). Unlike skeletal muscle, cardiac muscle cannot sustain anaerobic metabolism. Balancing oxygen demand with availability is crucial to cardiac function and survival, and regulated gene expression is a critical element of maintaining this balance. We investigated the role of the hypoxia‐inducible transcription factor HIF‐1α in maintaining this balance under normoxic conditions. Cardiac myocyte‐specific HIF‐ 1α gene deletion in the hearts of genetically engineered mice caused reductions in contractility, vascularization, high‐energy phosphate content, and lactate production. This was accompanied by altered calcium flux and altered expression of genes involved in calcium handling, angiogenesis, and glucose metabolism. These findings support a central role for HIF‐1α in coordinating energy availability and utilization in the heart and have implications for disease states in which cardiac oxygen delivery is impaired.

Loss of Skeletal Muscle HIF-1α Results in Altered Exercise Endurance

The physiological flux of oxygen is extreme in exercising skeletal muscle. Hypoxia is thus a critical parameter in muscle function, influencing production of ATP, utilization of energy-producing substrates, and manufacture of exhaustion-inducing metabolites. Glycolysis is the central source of anaerobic energy in animals, and this metabolic pathway is regulated under low-oxygen conditions by the transcription factor hypoxia-inducible factor 1α (HIF-1α). To determine the role of HIF-1α in regulating skeletal muscle function, we tissue-specifically deleted the gene encoding the factor in skeletal muscle. Significant exercise-induced changes in expression of genes are decreased or absent in the skeletal-muscle HIF-1α knockout mice (HIF-1α KOs); changes in activities of glycolytic enzymes are seen as well. There is an increase in activity of rate-limiting enzymes of the mitochondria in the muscles of HIF-1α KOs, indicating that the citric acid cycle and increased fatty acid oxidation may be compensating for decreased flow through the glycolytic pathway. This is corroborated by a finding of no significant decreases in muscle ATP, but significantly decreased amounts of lactate in the serum of exercising HIF-1α KOs. This metabolic shift away from glycolysis and toward oxidation has the consequence of increasing exercise times in the HIF-1α KOs. However, repeated exercise trials give rise to extensive muscle damage in HIF-1α KOs, ultimately resulting in greatly reduced exercise times relative to wild-type animals. The muscle damage seen is similar to that detected in humans in diseases caused by deficiencies in skeletal muscle glycogenolysis and glycolysis. Thus, these results demonstrate an important role for the HIF-1 pathway in the metabolic control of muscle function.

Hypoxia-Inducible Factor-Dependent Degeneration, Failure, and Malignant Transformation of the Heart in the Absence of the von Hippel-Lindau Protein

ABSTRACT Hypoxia-inducible transcription factor 1 (HIF-1) and HIF-2α regulate the expression of an expansive array of genes associated with cellular responses to hypoxia. Although HIF-regulated genes mediate crucial beneficial short-term biological adaptations, we hypothesized that chronic activation of the HIF pathway in cardiac muscle, as occurs in advanced ischemic heart disease, is detrimental. We generated mice with cardiac myocyte-specific deletion of the von Hippel-Lindau protein (VHL), an essential component of an E3 ubiquitin ligase responsible for suppressing HIF levels during normoxia. These mice were born at expected frequency and thrived until after 3 months postbirth, when they developed severe progressive heart failure and premature death. VHL-null hearts developed lipid accumulation, myofibril rarefaction, altered nuclear morphology, myocyte loss, and fibrosis, features seen for various forms of human heart failure. Further, nearly 50% of VHL−/− hearts developed malignant cardiac tumors with features of rhabdomyosarcoma and the capacity to metastasize. As compelling evidence for the mechanistic contribution of HIF-1α, the concomitant deletion of VHL and HIF-1α in the heart prevented this phenotype and restored normal longevity. These findings strongly suggest that chronic activation of the HIF pathway in ischemic hearts is maladaptive and contributes to cardiac degeneration and progression to heart failure.

Corticotropin-releasing factor receptor 2 is a tonic suppressor of vascularization

Angiogenesis is regulated by means of a balance between activators and inhibitors. However, little is known regarding the regulation of the quiescent state of adult vessels. Corticotropin-releasing factor receptor 2 (CRFR2) is found in both endothelial and smooth muscle cells (SMCs) in the vasculature, where its function has remained elusive. We have investigated the role of CRFR2 as a determinant of tissue vascularization by comparing control and CRFR2-deficient mice with immunohistological and morphometric techniques. To define the mechanisms responsible for CRFR2 inhibition of angiogenesis, we have also examined in vitro the effect of ligand activation on cell proliferation, cell cycle protein phosphorylation, and capillary tube formation. Our results demonstrate that mice deficient for CRFR2 become hypervascularized postnatally. Activation of this receptor in vitro results in reduced vascular endothelial growth factor (VEGF) release from SMCs, an inhibition of SMC proliferation, and inhibition of capillary tube formation in collagen gels. Treatment of a subcutaneously injected gel matrix with a CRFR2 agonist inhibits growth factor-induced vascularization. Western blots show that cell cycle retinoblastoma protein, which is essential for cell cycle progression, is decreased by CRFR2 agonist treatment in SMCs. These results suggest that CRFR2 is a critical component of a pathway necessary for tonic inhibition of adult neovascularization. CRFR2 may be a potential target for therapeutic modulation of angiogenesis in cancer and ischemic cardiovascular disease.

Decorin inhibition of PDGF-stimulated vascular smooth muscle cell function: potential mechanism for inhibition of intimal hyperplasia after balloon angioplasty.

Decorin is a small proteoglycan that binds to transforming growth factor-beta (TGF-beta) and inhibits its activity. However, its interaction with platelet-derived growth factor (PDGF), involved in arterial repair after injury, is not well characterized. The objectives of this study were to assess decorin-PDGF and decorin-PDGF receptor (PDGFR) interactions, the in vitro effects of decorin on PDGF-stimulated smooth muscle cell (SMC) functions and the in vivo effects of decorin overexpression on arterial repair in a rabbit carotid balloon-injury model. Decorin binding to PDGF was demonstrated by solid-phase binding and affinity cross-linking assays. Decorin potently inhibited PDGF-stimulated PDGFR phosphorylation. Pretreatment of rabbit aortic SMC with decorin significantly inhibited PDGF-stimulated cell migration, proliferation, and collagen synthesis. Decorin overexpression by adenoviral-mediated gene transfection in balloon-injured carotid arteries significantly decreased intimal cross-sectional area and collagen content by approximately 50% at 10 weeks compared to beta-galactosidase-transfected or balloon-injured, non-transfected controls. This study shows that decorin binds to PDGF and inhibits its stimulatory activity on SMCs by preventing PDGFR phosphorylation. Decorin overexpression reduces intimal hyperplasia and collagen content after arterial injury. Decorin may be an effective therapy for the prevention of intimal hyperplasia after balloon angioplasty.

An engineered VEGF-activating zinc finger protein transcription factor improves blood flow and limb salvage in advanced-age mice

Advances in understanding the relationship between protein structure and DNA binding specificity have made it possible to engineer zinc finger protein (ZFP) transcription factors to specifically activate or repress virtually any gene. To evaluate the potential clinical utility of this approach for peripheral vascular disease, we investigated the ability of an engineered vascular endothelial growth factor (VEGFa)‐activating ZFP (MVZ+426b) to induce angiogenesis and rescue hindlimb ischemia in a murine model. Hindlimb ischemia was surgically induced in advanced‐age C57/BL6 mice. Adenovirus (Ad) encoding either MVZ+426b or the fluorescent marker dsRed was delivered to the adducter muscle of the ischemic hindlimb, and the effects on blood flow, limb salvage, and vascularization were assessed. Ad‐MVZ+426b induced expression of VEGFa at the mRNA and protein levels and stimulated a significant increase in vessel counts in the ischemic limb. This was accompanied by significantly increased blood flow and limb salvage as measured serially for 4 wk. These data demonstrate that activation of the endogenous VEGFa gene by an engineered ZFP can induce angiogenesis in a clinically relevant model and further document the feasibility of designing ZFPs to therapeutically regulate gene expression in vivo.