Background— Peripheral artery disease (PAD) is a prevalent cardiovascular disorder that results in tissue ischemia which can progress to critical limb ischemia. Restoration of tissue perfusion in the setting of chronic ischemia through stimulation of arteriogenesis and angiogenesis remains a key therapeutic target for PAD. However, experimental therapeutics, including growth factor and gene therapy, have had little clinical success indicating the need for a better understanding of molecular pathways required for therapeutic angiogenesis. Methods and Results— Here we report that phosphodiesterase-5 inhibition by sildenafil significantly increases vascular perfusion, tissue blood flow, and vascular density during chronic ischemia of the mouse hind limb. Importantly, sildenafil therapy did not alter any of these parameters in nonischemic limbs. Sildenafil increased tissue cGMP levels independently of increases in nitric oxide production, and sildenafil therapy stimulated angiogenesis in ischemic limbs of eNOS −/− and iNOS −/− mice. Lastly, sildenafil-mediated angiogenic activity was blocked by inhibition of protein kinase G using the PKG antagonist DT-3. Conclusions— These data demonstrate that sildenafil therapy results in increased angiogenic activity through a PKG-dependent pathway that is independent of nitric oxide production or NOS activity and identify the angiogenic therapeutic potential of sildenafil for critical limb ischemia.

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Previous studies suggest that inflammatory cell adhesion molecules may modulate endothelial cell migration and angiogenesis through unknown mechanisms. Using a combination of in vitro and in vivo approaches, herein we reveal a novel redox sensitive mechanism by which ICAM-1 modulates endothelial GSH that controls VEGF-A induced eNOS activity, endothelial chemotaxis, and angiogenesis. In vivo disk angiogenesis assays showed attenuated VEGF-A mediated angiogenesis in ICAM-1 −/− mice. Moreover, VEGF-A dependent chemotaxis, eNOS phosphorylation, and nitric oxide (NO) production were impaired in ICAM-1 −/− MAEC compared to WT MAEC. Decreasing intracellular GSH in ICAM-1 −/− MAEC to levels observed in WT MAEC with 150 μM buthionine sulfoximine (BSO) restored VEGF-A responses. Conversely, GSH supplementation of WT MAEC with 5 mM glutathione ethyl ester (GEE) mimicked defects observed in ICAM-1 −/− cells. Deficient angiogenic responses in ICAM-1 −/− cells were associated with increased expression of the lipid phosphatase, PTEN, consistent with antagonism of signaling pathways leading to eNOS activation. PTEN expression was also sensitive to GSH status, decreasing or increasing in proportion to intracellular GSH concentrations. These data suggest a novel role for ICAM-1 in modulating VEGF-A induced angiogenesis and eNOS activity through regulation of PTEN expression via modulation of intracellular GSH status.

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Two technical hurdles, gene delivery and target specificity, have hindered the development of effective cancer gene therapies. In order to circumvent the problem of tumor specificity, the suicide gene, HSV-1 thymidine kinase (HSV-Tk), was modified with a complex 5' upstream-untranslated region (5'-UTR) that limits efficient translation to cells expressing high levels of the translation initiation factor, eIF4E. Since previous studies have shown that most tumor cells express elevated levels of eIF4E, tumor-specific gene delivery was optimized by incorporation of the 5'-UTR-modified suicide gene (HSV-UTk) into an adenovirus vector (Ad-CMV-UTk). The efficacy of this novel approach of targeting suicide gene expression and limiting cytotoxicity by means of translational restriction was tested in vitro with the use of the human breast cancer cell lines (MCF-7, MDA-MB435, and ZR-75-1). As controls, normal MCF10A, HMEC, and HMSC cell lines that express relatively low levels of eIF4E were used. Real-time reverse-transcription polymerase chain reaction (RT-PCR) was used to quantify HSV-Tk mRNA for cells infected with Ad-CMV-UTk as well as with Ad-CMV-Tk (a control adenovirus in which HSV-Tk is not regulated at the level of translation). Translation of HSV-Tk in the Ad-infected cells was measured by Western blot analysis. In addition, cytotoxicity was determined following treatment with the pro-drug ganciclovir (GCV) using an MTT viability assay. Finally, microPET imaging was used to assess cancer cell-specific expression of HSV-Tk and expression in normal tissues in vivo after intraperitoneal injection of Ad-CMV-Tk or Ad-CMV-UTk. These data collectively showed enhanced cancer cell-specific gene expression and reduced normal tissue gene expression for the Ad-HSV-UTk compared to the Ad-CMV-Tk, leading to increased cancer cell-enhanced GCV cytotoxicity. These results indicate that translational targeting of suicide gene expression in tumor cells in vitro and in vivo is effective and may provide a platform for enhanced cancer gene therapy specificity.

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Although improvements in timing and approach for early reperfusion with acute coronary syndromes have occurred, myocardial injury culminating in a myocardial infarction (MI) remains a common event. Although a multifactorial process, an imbalance between the induction of proteolytic pathways, such as matrix metalloproteinases (MMPs) and endogenous tissue inhibitors of metalloproteinase (TIMPs), has been shown to contribute to this process. In the present study, a full-length TIMP-3 recombinant protein (rTIMP-3) was encapsulated in a specifically formulated hyaluronic acid (HA)-based hydrogel that contained MMP-cleavable peptide cross-links, which influenced the rate of rTIMP-3 release from the HA gel. The effects of localized delivery of this MMP-sensitive HA gel (HAMMPS) alone and containing rTIMP-3 (HAMMPS/rTIMP-3) were examined in terms of the natural history of post-MI remodeling. Pigs were randomized to one of the following three different groups: MI and saline injection (MI/saline group, 100-μl injection at nine injection sites, n = 7), MI and HAMMPS injection (MI/HAMMPS group; 100-μl injection at nine injection sites, n = 7), and MI and HAMMPS/rTIMP-3 injection (MI/HAMMPS/rTIMP-3 group; 20-μg/100-μl injection at nine injection sites, n = 7). Left ventricular (LV) echocardiography was serially performed up to 28 days post-MI. LV dilation, as measured by end-diastolic volume, and the degree of MI wall thinning were reduced by ~50% in the HAMMPS/rTIMP-3 group ( P < 0.05). Furthermore, indexes of heart failure progression post-MI, such as LV filling pressures and left atrial size, were also attenuated to the greatest degree in the HAMMPS/rTIMP-3 group. At 28 days post-MI, HAMMPS/rTIMP-3 caused a relative reduction in the transcriptional profile for myofibroblasts as well as profibrotic pathways, which was confirmed by subsequent histochemistry. In conclusion, these findings suggest that localized delivery of a MMP-sensitive biomaterial that releases a recombinant TIMP holds promise as a means to interrupt adverse post-MI remodeling. NEW & NOTEWORTHY The present study targeted a myocardial matrix proteolytic system, matrix metalloproteinases (MMPs), through the use of a recombinant tissue inhibitor of MMPs incorporated into a MMP-sensitive hydrogel, which was regionally injected using a large animal model of myocardial infarction. Left ventricular geometry and function and indexes of myocardial remodeling were improved with this approach and support the advancement of localized therapeutic strategies that specifically target the myocardial matrix.

OBJECTIVE—Diabetogenic T-cell recruitment into pancreatic islets faciltates β-cell destruction during autoimmune diabetes, yet specific mechanisms governing this process are poorly understood. The chemokine stromal cell–derived factor-1 (SDF-1) controls T-cell recruitment, and genetic polymorphisms of SDF-1 are associated with early development of type 1 diabetes. RESEARCH DESIGN AND METHODS—Here, we examined the role of SDF-1 regulation of diabetogenic T-cell adhesion to islet microvascular endothelium. Islet microvascular endothelial cell monolayers were activated with tumor necrosis factor-α (TNF-α), subsequently coated with varying concentrations of SDF-1 (1–100 ng/ml), and assayed for T-cell/endothelial cell interactions under physiological flow conditions. RESULTS—TNF-α significantly increased NOD/LtJ T-cell adhesion, which was completely blocked by SDF-1 in a dose-dependent manner, revealing a novel chemorepulsive effect. Conversely, SDF-1 enhanced C57BL/6J T-cell adhesion to TNF-α–activated islet endothelium, demonstrating that SDF-1 augments normal T-cell adhesion. SDF-1 chemorepulsion of NOD/LtJ T-cell adhesion was completely reversed by blocking Giα-protein–coupled receptor activity with pertussis toxin. CXCR4 protein expression was significantly decreased in NOD/LtJ T-cells, and inhibition of CXCR4 activity significantly reversed SDF-1 chemorepulsive effects. Interestingly, SDF-1 treatment significantly abolished T-cell resistance to shear-mediated detachment without altering adhesion molecule expression, thus demonstrating decreased integrin affinity and avidity. CONCLUSIONS—In this study, we have identified a previously unknown novel function of SDF-1 in negatively regulating NOD/LtJ diabetogenic T-cell adhesion, which may be important in regulating diabetogenic T-cell recruitment into islets.