Frank J. Giordano

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.

The VIVA Trial

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 · kg−1 · min−1), or high-dose rhVEGF (50 ng · kg−1 · 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.

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.

Effect of Intracoronary Recombinant Human Vascular Endothelial Growth Factor on Myocardial Perfusion Evidence for a Dose-Dependent Effect

BACKGROUND Animal models of therapeutic angiogenesis have stimulated development of clinical application in patients with limited options for coronary revascularization. The impact of recombinant human vascular endothelial growth factor (rhVEGF) on myocardial perfusion in humans has not been reported. METHODS AND RESULTS Fourteen patients underwent exercise (n=11), dobutamine (n=2), or dipyridamole (n=1) myocardial perfusion single photon emission CT (SPECT) before as well as 30 and 60 days after rhVEGF administration. After uniform processing and display, 2 observers blinded to the timing of the study and dose of rhVEGF reviewed the SPECT images. By a visual, semiquantitative 20-segment scoring method, summed stress scores (SSS) and summed rest scores (SRS) were generated. Although the SSS did not change from baseline to 30 days (21.6 versus 21.5; P=NS), the SRS improved after rhVEGF (13.2 versus 10.4; P<0.05). Stress and rest perfusion improved in >2 segments infrequently in patients treated with low-dose rhVEGF. However, 5 of 6 patients had improvement in >2 segments at rest and stress with the higher rhVEGF doses. Furthermore, although neither the SSS nor the SRS changed in patients treated with the low doses, the SRS decreased in the high-dose rhVEGF patients at 60 days (14.7 versus 10.7; P<0.05). Quantitative analysis was consistent with the visual findings but failed to demonstrate statistical significance. CONCLUSIONS Although not designed to demonstrate rhVEGF efficacy, these phase 1 data support the concept that rhVEGF improves myocardial perfusion at rest and provide evidence of a dose-dependent effect.

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.

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.

Decreased expression of the DNA mismatch repair gene Mlh1 under hypoxic stress in mammalian cells

ABSTRACT The hypoxic tumor microenvironment has been shown to contribute to genetic instability. As one possible mechanism for this effect, we report that expression of the DNA mismatch repair (MMR) gene Mlh1 is specifically reduced in mammalian cells under hypoxia, whereas expression of other MMR genes, including Msh2, Msh6, and Pms2, is not altered at the mRNA level. However, levels of the PMS2 protein are reduced, consistent with destabilization of PMS2 in the absence of its heterodimer partner, MLH1. The hypoxia-induced reduction in Mlh1 mRNA was prevented by the histone deacetylase inhibitor trichostatin A, suggesting that hypoxia causes decreased Mlh1 transcription via histone deacetylation. In addition, treatment of cells with the iron chelator desferrioxamine also reduced MLH1 and PMS2 levels, in keeping with low oxygen tension being the stress signal that provokes the altered MMR gene expression. Functional MMR deficiency under hypoxia was detected as induced instability of a (CA)29 dinucleotide repeat and by increased mutagenesis in a chromosomal reporter gene. These results identify a potential new pathway of genetic instability in cancer: hypoxia-induced reduction in the expression of key MMR proteins. In addition, this stress-induced genetic instability may represent a conceptual parallel to the pathway of stationary-phase mutagenesis seen in bacteria.

VEGF Receptor 2 Endocytic Trafficking Regulates Arterial Morphogenesis

VEGF is the key growth factor regulating arterial morphogenesis. However, molecular events involved in this process have not been elucidated. Synectin null mice demonstrate impaired VEGF signaling and a marked reduction in arterial morphogenesis. Here, we show that this occurs due to delayed trafficking of VEGFR2-containing endosomes that exposes internalized VEGFR2 to selective dephosphorylation by PTP1b on Y(1175) site. Synectin involvement in VEGFR2 intracellular trafficking requires myosin-VI, and myosin-VI knockout in mice or knockdown in zebrafish phenocopy the synectin null phenotype. Silencing of PTP1b restores VEGFR2 activation and significantly recovers arterial morphogenesis in myosin-VI(-/-) knockdown zebrafish and synectin(-/-) mice. We conclude that activation of the VEGF-mediated arterial morphogenesis cascade requires phosphorylation of the VEGFR2 Y(1175) site that is dependent on trafficking of internalized VEGFR2 away from the plasma membrane via a synectin-myosin-VI complex. This key event in VEGF signaling occurs at an intracellular site and is regulated by a novel endosomal trafficking-dependent process.