Dinggang Liu

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.

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.

Renalase deficiency aggravates ischemic myocardial damage

Chronic kidney disease (CKD) leads to an 18-fold increase in cardiovascular complications not fully explained by traditional risk factors. Levels of renalase, a recently discovered oxidase that metabolizes catecholamines, are decreased in CKD. Here we show that renalase deficiency in a mouse knockout model causes increased plasma catecholamine levels and hypertension. Plasma blood urea nitrogen, creatinine, and aldosterone were unaffected. However, knockout mice had normal systolic function and mild ventricular hypertrophy but tolerated cardiac ischemia poorly and developed myocardial necrosis threefold more severe than that found in wild-type mice. Treatment with recombinant renalase completely rescued the cardiac phenotype. To gain insight into the mechanisms mediating this cardioprotective effect, we tested if gene deletion affected nitrate and glutathione metabolism, but found no differences between hearts of knockout and wild-type mice. The ratio of oxidized (NAD) to reduced (NADH) nicotinamide adenine dinucleotide in cardiac tissue, however, was significantly decreased in the hearts of renalase knockout mice, as was plasma NADH oxidase activity. In vitro studies confirmed that renalase metabolizes NADH and catecholamines. Thus, renalase plays an important role in cardiovascular pathology and its replacement may reduce cardiac complications in renalase-deficient states such as CKD.

Endothelial Expression of β1 Integrin Is Required for Embryonic Vascular Patterning and Postnatal Vascular Remodeling

ABSTRACT The largest subgroup of integrins is that containing the β1 subunit. β1 integrins have been implicated in a wide array of biological processes ranging from adhesion to cell growth, organogenesis, and mechanotransduction. Global deletion of β1 integrin expression results in embryonic death at ca. embryonic day 5 (E5), a developmental time point too early to determine the effects of this integrin on vascular development. To elucidate the specific role of β1 integrin in the vasculature, we conditionally deleted the β1 gene in the endothelium. Homozygous deletion of β1 integrins in the endothelium resulted in failure of normal vascular patterning, severe fetal growth retardation, and embryonic death at E9.5 to 10, although there were no overt effects on vasculogenesis. Heterozygous endothelial β1 gene deletion did not diminish fetal or postnatal survival, but it reduced β1 subunit expression in endothelial cells from adult mice by approximately 40%. These mice demonstrated abnormal vascular remodeling in response to experimentally altered in vivo blood flow and diminished vascularization in healing wounds. These data demonstrate that endothelial expression of β1 integrin is required for developmental vascular patterning and that endothelial β1 gene dosing has significant functional effects on vascular remodeling in the adult. Understanding how β1 integrin expression is modulated may have significant clinical importance.

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.

Normal glucose uptake in the brain and heart requires an endothelial cell-specific HIF-1α-dependent function.

Although intimately positioned between metabolic substrates in the bloodstream and the tissue parenchymal cells that require these substrates, a major role of the vascular endothelium in the regulation of tissue metabolism has not been widely appreciated. We hypothesized that via control of transendothelial glucose transport and contributing paracrine mechanisms the endothelium plays a major role in regulating organ and tissue glucose metabolism. We further hypothesized that the hypoxia-inducible factor -1α (HIF-1α) plays an important role in coordinating these endothelial functions. To test these hypotheses, we generated mice with endothelial cell-specific deletion of HIF-1α. Loss of HIF in the endothelium resulted in significantly increased fasting blood glucose levels, a blunted insulin response with delayed glucose clearance from the blood after i.v. loading, and significantly decreased glucose uptake into the brain and heart. Endothelial HIF-1α knockout mice also exhibited a reduced cerebrospinal fluid/blood glucose ratio, a finding consistent with reduced transendothelial glucose transport and a diagnostic criterion for the Glut1 deficiency genetic syndrome. Endothelial cells from these mice demonstrated decreased Glut1 levels and reduced glucose uptake that was reversed by forced expression of Glut1. These data strongly support an important role of the vascular endothelium in determining whole-organ glucose metabolism and indicate that HIF-1α is a critical mediator of this function.

Smooth Muscle Hypoxia-Inducible Factor 1α Links Intravascular Pressure and Atherosclerosis

Objective— We hypothesized that the hypoxia-inducible factor (HIF) 1&agr; in vascular smooth muscle contributes to the development of atherosclerosis, and links intravascular pressure to this process. Approach and Results— Transverse aortic constriction was used to create high-pressure vascular segments in control, apolipoprotein E (ApoE)−/−, smooth muscle-HIF1&agr;−/− , and ApoE−/− ×smooth muscle-HIF1&agr;−/− double-knockout mice. Transverse aortic constriction selectively induced atherosclerosis in high-pressure vascular segments in young ApoE−/− mice on normal chow, including coronary plaques within 1 month. Concomitant deletion of HIF1&agr; from smooth muscle significantly reduced vascular inflammation, and attenuated atherosclerosis. Conclusions— HIF1&agr; in vascular smooth muscle plays an important role in the pathogenesis of atherosclerosis, and may provide a mechanistic link between blood pressure, vascular inflammation, and lipid deposition.

Smooth Muscle Hypoxia-Inducible Factor 1α Links Intravascular Pressure and Atherosclerosis—Brief Report

Objective— We hypothesized that the hypoxia-inducible factor (HIF) 1&agr; in vascular smooth muscle contributes to the development of atherosclerosis, and links intravascular pressure to this process. Approach and Results— Transverse aortic constriction was used to create high-pressure vascular segments in control, apolipoprotein E (ApoE)−/−, smooth muscle-HIF1&agr;−/− , and ApoE−/− ×smooth muscle-HIF1&agr;−/− double-knockout mice. Transverse aortic constriction selectively induced atherosclerosis in high-pressure vascular segments in young ApoE−/− mice on normal chow, including coronary plaques within 1 month. Concomitant deletion of HIF1&agr; from smooth muscle significantly reduced vascular inflammation, and attenuated atherosclerosis. Conclusions— HIF1&agr; in vascular smooth muscle plays an important role in the pathogenesis of atherosclerosis, and may provide a mechanistic link between blood pressure, vascular inflammation, and lipid deposition.

43. Targeting the Biology of Heart Disease: Engineered Zinc Finger Protein Repressors of Phospholamban as a Potential Therapy for Congestive Heart Failure

Improper calcium handling of the heart is a hallmark of patients with congestive heart failure (CHF). Because calcium is critical for cardiac contractility, proteins that regulate calcium homeostasis are potential targets treating CHF. Phospholamban (PLN) decreases contractility by inhibiting the activity of Sarcoplasmic Reticulum Ca2+ ATPase 2 isoform A (SERCA2a); an increased PLN/SERCA2a ratio is often found in CHF patients. Recent studies have demonstrated that ablation or inhibition of PLN function can improve cardiac contractile properties in animal models of CHF, suggesting that down-regulation of PLN may improve cardiac function in CHF patients. Importantly, inhibition of PLN enhances calcium handling without activating b-adrenergic pathways, which is known to have many side-effects and increase mortality. The development of small-molecule inhibitors of PLN function has so far been unsuccessful, largely due to the difficulty of inhibiting protein-protein interactions (such as that between PLN and SERCA2a) using small molecules. On the other hand, approaches that aim to block the expression of PLN may provide a superior means of achieving the desired therapeutic effect.

936. Phage-Selected Motifs Internalize Via Multiple Endocytic Pathways and Facilitate Gene Delivery

Top of pageAbstract The ability to direct internalization of macromolecules into cells or across endothelial barriers efficiently has tremendous therapeutic implications. Several internalization motifs (IM) that exist in nature have been defined and exploited, including the third -helix of the Drosophila Antennapedia homeodomain and the HIV TAT protein. To investigate the general properties required for protein-based macromolecule internalization we constructed and performed peptide phage display (PPD) with T7 bacteriophage random 7-mer peptide libraries. Functional biopanning with the T7 libraries was performed on microvascular endothelial cells (EC) to select peptide motifs that could internalize phage. The titer of internalized phage increased through sequential rounds of biopanning and resulted in selection of phage expressing IM with a finite shared sequence relationship between charged, polar, and hydrophobic amino acids. Selected IM were synthesized as rhodamine-labeled free peptides and their internalization properties were investigated in EC. Internalization of IM was inhibited at 4 degrees, suggesting an energy dependent mechanism of internalization. Confocal data showed that the IM was internalized via at least two different endocytic pathways: clathrin-coated pits and lipid rafts. Treatment with inhibitors of clathrin-mediated and lipid raft endocytosis inhibited uptake of IM, consistent with the result from confocal study. Further, in vitro and in vivo studies indicated that precomplexing of adenovirus with the IM facilitated the degree of viral transduction into endothelial cells and across endothelial barriers. These data suggest that relatively short peptide motifs are capable of directing macromolecules to diverse internalization pathways. It may be possible to exploit these characteristics for targeted delivery of therapeutic macromolecules to cells and tissues, and potentially to develop more efficient methods of gene transfer.