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Biomedical subjects

Frank J Giordano

Publications and source records attributed to Frank J Giordano.

At least 19 recordsLinked to original sources
PubMed2006

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.

Adenoviridae↗
PubMed2005

Smad2 and Smad3 play different roles in rat hepatic stellate cell function and alpha-smooth muscle actin organization.

Hepatic stellate cells (HSC) play a central role in the pathogenesis of liver fibrosis, transdifferentiating in chronic liver disease from "quiescent" HSC to fibrogenic myofibroblasts. Transforming growth factor-beta (TGF-beta), acting both directly and indirectly, is a critical mediator of this process. To characterize the function of the TGF-beta signaling intermediates Smad2 and Smad3 in HSC, we infected primary rat HSC in culture with adenoviruses expressing wild-type and dominant negative Smads 2 and 3. Smad3-overexpressing cells exhibited increased deposition of fibronectin and type 1 collagen, increased chemotaxis, and decreased proliferation compared with uninfected cells and those infected with Smad2 or either dominant negative, demonstrating different biological functions for the two Smads. Additionally, coinfection experiments suggested that Smad2 and Smad3 signal via independent pathways. Smad3-overexpressing cells as well as TGF-beta-treated cells demonstrated more focal adhesions and increased alpha-smooth muscle actin (alpha-SMA) organization in stress fibers, although all cells reached the same level of alpha-SMA expression, indicating that Smad3 also regulates cytoskeletal organization in HSC. We suggest that TGF-beta, signaling via Smad3, plays an important role in the morphological and functional maturation of hepatic myofibroblasts.

Actins↗
PubMed2005

Oxygen, oxidative stress, hypoxia, and heart failure.

A constant supply of oxygen is indispensable for cardiac viability and function. However, the role of oxygen and oxygen-associated processes in the heart is complex, and they and can be either beneficial or contribute to cardiac dysfunction and death. As oxygen is a major determinant of cardiac gene expression, and a critical participant in the formation of ROS and numerous other cellular processes, consideration of its role in the heart is essential in understanding the pathogenesis of cardiac dysfunction.

Animals↗
PubMed2004

Stromal cell-derived factor-1alpha 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-1alpha (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. CONCLUSIONS: 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.

Animals↗
PubMed2004

Loss of skeletal muscle HIF-1alpha 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 1alpha (HIF-1alpha). To determine the role of HIF-1alpha 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-1alpha knockout mice (HIF-1alpha 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-1alpha 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-1alpha KOs. This metabolic shift away from glycolysis and toward oxidation has the consequence of increasing exercise times in the HIF-1alpha KOs. However, repeated exercise trials give rise to extensive muscle damage in HIF-1alpha 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.

Adenosine Triphosphate↗
PubMed2004

Cardiac myocyte-specific HIF-1alpha 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-1alpha in maintaining this balance under normoxic conditions. Cardiac myocyte-specific HIF-1alpha 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-1alpha in coordinating energy availability and utilization in the heart and have implications for disease states in which cardiac oxygen delivery is impaired. Heart muscle requires a constant supply of oxygen. When oxygen supply does not match myocardial demand cardiac contractile dysfunction occurs, and prolongation of this mismatch leads to apoptosis and necrosis. Coordination of oxygen supply and myocardial demand involves immediate adaptations, such as coronary vasodilatation, and longer-term adaptations that include altered patterns of gene expression (2-4). How the expression of multiple genes is coordinated with oxygen availability in the heart and the impact of oxygen-dependent gene expression on cardiac function are insufficiently understood. Further elucidating these relationships may help clarify the molecular pathology of various cardiovascular disease states, including ischemic cardiomyopathy and myocardial hibernation (5, 6).

Animals↗
PubMed2004

Loss of HIF-1alpha in endothelial cells disrupts a hypoxia-driven VEGF autocrine loop necessary for tumorigenesis.

We deleted the hypoxia-responsive transcription factor HIF-1alpha in endothelial cells (EC) to determine its role during neovascularization. We found that loss of HIF-1alpha inhibits a number of important parameters of EC behavior during angiogenesis: these include proliferation, chemotaxis, extracellular matrix penetration, and wound healing. Most strikingly, loss of HIF-1alpha in EC results in a profound inhibition of blood vessel growth in solid tumors. These phenomena are all linked to a decreased level of VEGF expression and loss of autocrine response of VEGFR-2 in HIF-1alpha null EC. We thus show that a HIF-1alpha-driven, VEGF-mediated autocrine loop in EC is an essential component of solid tumor angiogenesis.

Animals↗
PubMed2004

AMP-activated protein kinase mediates ischemic glucose uptake and prevents postischemic cardiac dysfunction, apoptosis, and injury.

AMP-activated protein kinase (AMPK) is an important regulator of diverse cellular pathways in the setting of energetic stress. Whether AMPK plays a critical role in the metabolic and functional responses to myocardial ischemia and reperfusion remains uncertain. We examined the cardiac consequences of long-term inhibition of AMPK activity in transgenic mice expressing a kinase dead (KD) form of the enzyme. The KD mice had normal fractional shortening and no heart failure, cardiac hypertrophy, or fibrosis, although the in vivo left ventricular (LV) dP/dt was lower than that in WT hearts. During low-flow ischemia and postischemic reperfusion in vitro, KD hearts failed to augment glucose uptake and glycolysis, although glucose transporter content and insulin-stimulated glucose uptake were normal. KD hearts also failed to increase fatty acid oxidation during reperfusion. Furthermore, KD hearts demonstrated significantly impaired recovery of LV contractile function during postischemic reperfusion that was associated with a lower ATP content and increased injury compared with WT hearts. Caspase-3 activity and TUNEL-staining were increased in KD hearts after ischemia and reperfusion. Thus, AMPK is responsible for activation of glucose uptake and glycolysis during low-flow ischemia and plays an important protective role in limiting damage and apoptotic activity associated with ischemia and reperfusion in the heart.

AMP-Activated Protein Kinases↗
PubMed2004

Noninvasive imaging of myocardial angiogenesis following experimental myocardial infarction.

Noninvasive imaging strategies will be critical for defining the temporal characteristics of angiogenesis and assessing efficacy of angiogenic therapies. The alphavbeta3 integrin is expressed in angiogenic vessels and represents a potential novel target for imaging myocardial angiogenesis. We demonstrated the localization of an indium-111-labeled ((111)In-labeled) alphavbeta3-targeted agent in the region of injury-induced angiogenesis in a chronic rat model of infarction. The specificity of the targeted alphavbeta3-imaging agent for angiogenesis was established using a nonspecific control agent. The potential of this radiolabeled alphavbeta3-targeted agent for in vivo imaging was then confirmed in a canine model of postinfarction angiogenesis. Serial in vivo dual-isotope single-photon emission-computed tomographic (SPECT) imaging with the (111)In-labeled alphavbeta3-targeted agent demonstrated focal radiotracer uptake in hypoperfused regions where angiogenesis was stimulated. There was a fourfold increase in myocardial radiotracer uptake in the infarct region associated with histological evidence of angiogenesis and increased expression of the alphavbeta3 integrin. Thus, angiogenesis in the heart can be imaged noninvasively with an (111)In-labeled alphavbeta3-targeted agent. The noninvasive evaluation of angiogenesis may have important implications for risk stratification of patients following myocardial infarction. This approach may also have significant clinical utility for noninvasively tracking therapeutic myocardial angiogenesis.

Animals↗
PubMed2003

The VIVA trial: Vascular endothelial growth factor in Ischemia for Vascular Angiogenesis.

BACKGROUND: Recombinant human vascular endothelial growth factor protein (rhVEGF) stimulates angiogenesis in animal models and was well tolerated in Phase I clinical trials. VIVA (Vascular endothelial growth factor in Ischemia for Vascular Angiogenesis) is a double-blind, placebo-controlled trial designed to evaluate the safety and efficacy of intracoronary and intravenous infusions of rhVEGF. METHODS AND RESULTS: A total of 178 patients with stable exertional angina, unsuitable for standard revascularization, were randomized to receive placebo, low-dose rhVEGF (17 ng x kg(-1) x min(-1)), or high-dose rhVEGF (50 ng x kg(-1) x min(-1)) by intracoronary infusion on day 0, followed by intravenous infusions on days 3, 6, and 9. Exercise treadmill tests, angina class, and quality of life assessments were performed at baseline, day 60, and day 120. Myocardial perfusion imaging was performed at baseline and day 60. At day 60, the change in exercise treadmill test (ETT) time from baseline was not different between groups (placebo, +48 seconds; low dose, +30 seconds; high dose, +30 seconds). Angina class and quality of life were significantly improved within each group, with no difference between groups. By day 120, placebo-treated patients demonstrated reduced benefit in all three measures, with no significant difference compared with low-dose rhVEGF. In contrast, high-dose rhVEGF resulted in significant improvement in angina class (P=0.05) and nonsignificant trends in ETT time (P=0.15) and angina frequency (P=0.09) as compared with placebo. CONCLUSIONS: rhVEGF seems to be safe and well tolerated. rhVEGF offered no improvement beyond placebo in all measurements by day 60. By day 120, high-dose rhVEGF resulted in significant improvement in angina and favorable trends in ETT time and angina frequency.

Adult↗
PubMed2003

Cell-permeable peptides improve cellular uptake and therapeutic gene delivery of replication-deficient viruses in cells and in vivo.

Small polybasic peptides derived from the transduction domains of certain proteins, such as the third alpha-helix of the Antennapedia (Antp) homeodomain, can cross the cell membrane through a receptor-independent mechanism. These cell-permeable molecules have been used as 'Trojan horses' to introduce biologically active cargo molecules such as DNA, peptides or proteins into cells. Using these cell-permeable peptides, we have developed an efficient and simple method to increase virally mediated gene delivery and protein expression in vitro and in vivo. Here, we show that cell-permeable peptides increase viral cell entry, improve gene expression at reduced titers of virus and improve efficacy of therapeutically relevant genes in vivo.

Adenoviridae↗
PubMed2003

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.

Angioplasty, Balloon↗
PubMed2003

Gene-based therapeutic angiogenesis.

Stimulating new blood vessel growth in ischemic hearts or limbs is a hopeful new approach for patients with advanced vascular disease. This approach is based generally upon the hypothesis that sufficient exposure of a vascular bed to an angiogenic protein will stimulate neovascularization. Most angiogenic proteins have a markedly short serum half-life. To overcome this, researchers have turned to gene therapy to ensure continuous expression of angiogenic proteins and prolonged exposure in the targeted vascular beds. This field is still evolving, and although early clinical trial results suggest angiogenic gene therapy can be successful, many questions remain. As we continue to learn more about the complex interplay and coordinated action of the various factors involved in regulating angiogenesis, it is likely that strategies for therapeutic angiogenesis will continue to change. This review addresses the current state of angiogenic gene therapy, contrasts gene therapy with angiogenic protein delivery, describes early and recent clinical trial data, and discusses potential new directions in the field.

Angiogenesis Inducing Agents↗
PubMed2003

A new role for corticotropin-releasing factor receptor-2: suppression of vascularization.

The corticotropin-releasing factor (CRF) family of receptors and ligands are known to have potent effects on vasculature function. Our recent data has described a novel role of corticotropin-releasing factor receptor-2 (CRFR2) as a tonic inhibitor of neovascularization. CRFR2 is found in both endothelial and smooth muscle cells (SMC) in the vasculature, where its function has been elusive. From investigation into the role of CRFR2 as a determinant of tissue vascularization, it was discovered that mice deficient for CRFR2 become hypervascularized postnatally and express increased levels of vascular endothelial growth factor (VEGF). Mechanistically, CRFR2 activation in vitro was found to result in reduced VEGF release from SMCs, an inhibition of SMC proliferation, and an inhibition of capillary tube formation in collagen gels. Treatment of a subcutaneously injected gel matrix with a CRFR2 agonist inhibited growth factor-induced vascularization in vivo. Western blots for the cell-cycle retinoblastoma protein (Rb)--essential for cell-cycle progression, showed decreased levels of phosphorylated Rb following CRFR2 agonist treatment in SMCs--supporting a role for CRFR2 in regulation of SMC proliferation. These results suggest that CRFR2 is a critical component of a novel pathway necessary for tonic inhibition of adult neovascularization, and that CRFR2 may be a potential target for therapeutic modulation of angiogenesis.

Animals↗
PubMed2002

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 Vegfa(164) 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.

3T3 Cells↗
PubMed2002

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.

Angiogenesis Inhibitors↗
PubMed2002

Restoration of deficient membrane proteins in the cardiomyopathic hamster by in vivo cardiac gene transfer.

BACKGROUND: One of the most important problems in developing in vivo cardiac gene transfer has been low transfection efficiency. A novel in vivo technique was developed, tested in normal hamsters, and the feasibility of restoring a deficient structural protein (delta-sarcoglycan) in the cardiomyopathic (CM) hamster evaluated. METHODS AND RESULTS: Adenoviral (AdV) vectors encoding either the lacZ gene or delta-sarcoglycan gene were constructed. Hypothermia was achieved in hamsters by external body cooling to a rectal temperature of 18 to 25 degrees C. Through a small thoracotomy, the ascending aorta and the main pulmonary artery were occluded with snares, and cardioplegic solution containing histamine was injected into the aortic root; viral constructs were delivered 3 to 5 minutes later followed by release of the occluders and rewarming. Four days later, homogeneous beta-galactosidase expression was detected throughout the ventricles of the normal hearts (average 77.3+/-9.0% [SEM] of left ventricular myocytes). At 1 and 3 weeks after transfection, immunostaining showed extensive restoration of delta-sarcoglycan as well as alpha- and beta-sarcoglycan proteins to the myocyte membranes, despite loss of beta-galactosidase expression at 3 weeks. Also, at 3 weeks after gene transfer, there was significantly less progression of left ventricular dysfunction assessed as percent change in fractional shortening compared with controls. CONCLUSIONS: This study demonstrates the feasibility of high efficiency in vivo myocardial gene transfer and shows application in improving the level of a deficient cardiac structural protein and cardiac function in CM hamsters. The approach should be useful for assessing effects of expressing other genes that influence the structure or function of the normal and failing heart.

Adenoviridae↗