Perlecan heparan sulfate proteoglycan is a critical determinant of angiogenesis in response to mouse hind-limb ischemia.

BACKGROUND: Perlecan is a heparan sulfate proteoglycan (HSPG) constituent of the extracellular matrix with roles in cell growth, differentiation, and angiogenesis. The role of the HS side chains in regulating in vivo angiogenesis after hind-limb ischemia is unknown. METHODS: Heparan sulfate (HS)-deficient perlecan (Hspg2(Δ3/Δ3)) mice (n = 35), containing normal perlecan core protein but deficient in HS side chains, and wild-type (n = 33) littermates underwent surgical induction of hind-limb ischemia. Laser Doppler perfusion imaging (LDPI) and contrast-enhanced ultrasonography (CEU) provided serial assessment of hind-limb perfusion. Harvested muscles underwent immunostaining for endothelial cell density (CD31), real-time reverse transcription polymerase chain reaction RT-PCR for vascular endothelial growth factor (VEGF) mRNA expression and western blot analysis for VEGF and fibroblast growth factor (FGF)2 protein expression at days 2 and 28. RESULTS: Serial LDPI showed significantly greater perfusion recovery in ischemic limbs of wild-type compared with Hspg2(Δ3/Δ3) mice. CEU showed that normalized microvascular perfusion was increased in wild-type compared with Hspg2(Δ3/Δ3) mice at day 28 (0.67 ± 0.12 vs 0.26 ± 0.08; P = 0.001). CD31-positive cell counts were significantly higher in wild-type compared with Hspg2(Δ3/Δ3) mice on day 28 (122 ± 30 cells vs 84 ± 34 cells per high-power field [HPF]; P < 0.05). Endogenous VEGF mRNA expression (P < 0.05) and VEGF protein expression (P < 0.002) were significantly decreased in the ischemic limbs of Hspg2(Δ3/Δ3) mice compared with wild-type mice at day 2 and day 28, respectively. FGF2 protein expression showed no significant differences. CONCLUSIONS: These results suggest that the HS side chains in perlecan are important mediators of the angiogenic response to ischemia through a mechanism that involves upregulation of VEGF expression.

Graphene mechanical oscillators with tunable frequency.

Oscillators, which produce continuous periodic signals from direct current power, are central to modern communications systems, with versatile applications including timing references and frequency modulators. However, conventional oscillators typically consist of macroscopic mechanical resonators such as quartz crystals, which require excessive off-chip space. Here, we report oscillators built on micrometre-size, atomically thin graphene nanomechanical resonators, whose frequencies can be electrostatically tuned by as much as 14%. Self-sustaining mechanical motion is generated and transduced at room temperature in these oscillators using simple electrical circuitry. The prototype graphene voltage-controlled oscillators exhibit frequency stability and a modulation bandwidth sufficient for the modulation of radiofrequency carrier signals. As a demonstration, we use a graphene oscillator as the active element for frequency-modulated signal generation and achieve efficient audio signal transmission.

Tie2-dependent neovascularization of the ischemic hindlimb is mediated by angiopoietin-2.

The angiopoietins (ANGPT) are ligands for the endothelial cell (EC) receptor tyrosine kinase, Tie2. Angpt-1 is a Tie2 agonist that promotes vascular maturation and stabilization, whereas Angpt-2 is a partial agonist/antagonist involved in the initiation of postnatal angiogenesis. Therefore, we hypothesized that overexpression of Angpt-2 would be more effective than Angpt-1 for enhancing the perfusion recovery in the ischemic hindlimb. Perfusion recovery was markedly impaired in Tie2-deficient animals at day 35 in a model of chronic hindlimb ischemia. Injections of Angpt-2 or VEGFA plasmid at 7 days post femoral artery resection enhanced recovery and improved arteriogenesis as assessed by angiographic scores, whereas Angpt-1 or null plasmid had no effect. In addition, Angpt-2 together with VEGF resulted in greater improvement in perfusion and collateral vessel formation than VEGF alone. Similarly, conditional overexpression of Angpt-2 in mice improved ischemic limb blood flow recovery, while Angpt-1 overexpression was ineffective. These data from Tie2 heterozygote deficient mice demonstrate, for the first time, the importance of the Tie2 pathway in spontaneous neovascularization in response to chronic hindlimb ischemia. Moreover, they show that overexpression of the partial agonist, Angpt-2, but not Angpt-1, enhanced ischemic hind limb perfusion recovery and collateralization, suggesting that a coordinated sequence antagonist and agonist activity is required for effective therapeutic revascularization.

Therapeutic arteriogenesis by ultrasound-mediated VEGF165 plasmid gene delivery to chronically ischemic skeletal muscle.

Current methods of gene delivery for therapeutic angiogenesis are invasive, requiring either intraarterial or intramuscular administration. A noninvasive method of gene delivery has been developed using ultrasound-mediated destruction of intravenously administered DNA-bearing carrier microbubbles during their microcirculatory transit. Here we show that chronic ischemia could be markedly improved by ultrasound-mediated destruction of microbubbles bearing vascular endothelial growth factor-165 (VEGF(165)) plasmid DNA. Using a model of severe chronic hindlimb ischemia in rats, we demonstrated that ultrasound mediated VEGF(165)/green fluorescent protein (GFP) plasmid delivery resulted in a significant improvement in microvascular blood flow by contrast-enhanced ultrasound, and an increased vessel density by fluorescent microangiography, with minimal changes in control groups. The improvement in tissue perfusion was attributed predominantly to increases in noncapillary blood volume or arteriogenesis, with perfusion peaking at 14 days after delivery, followed by a partial regression of neovascularization at 6 weeks. Transfection was localized predominantly to the vascular endothelium of arterioles in treated ischemic muscle. RT-PCR confirmed the presence of VEGF(165)/GFP mRNA within treated ischemic muscle, being highest at day 3 postdelivery, and subsequently decreasing, becoming almost undetectable by 6 weeks. We found a modulation of endogenous growth factor expression in VEGF-treated ischemic muscle, consistent with a biologic effect of ultrasound mediated gene delivery. The results of our study demonstrate the utility of ultrasonic destruction of plasmid-bearing microbubbles to induce therapeutic arteriogenesis in the setting of severe chronic ischemia.

Growth factor-induced therapeutic neovascularization for ischaemic vascular disease: time for a re-evaluation?

PURPOSE OF REVIEW: Therapeutic angiogenesis and arteriogenesis represent an alternative treatment modality for patients with advanced ischaemic coronary or peripheral artery occlusive disease, who are unsuitable for standard revascularization procedures. RECENT DEVELOPMENTS: Proof-of-concept evidence for therapeutic growth factor, both gene and protein-mediated neovascularization was provided in animal models of chronic myocardial and hindlimb ischaemia. Early human, phase I, trials utilizing the prototypical growth factor families, vascular endothelial growth factor and fibroblast growth factor, documented safety and suggested improvements in anginal symptoms and functional status. Large, randomized, placebo-controlled phase II/III clinical trials have, however, yielded variable results as such studies have suffered from significant limitations in therapeutic approach or design, which limits the ability to draw firm conclusions. SUMMARY: Future trials must incorporate robust delivery strategies and address issues of study design including proper patient selection. Laboratory-based refinements in therapy, including a focus on the promotion of arteriogenesis and the modification of patient 'endotheliopathy', will all further enhance the potential of therapeutic neovascularization strategies.

Therapeutic neovascularization for ischemic heart disease.

The demonstration that angiogenic growth factors stimulate new blood vessel growth and restore perfusion in animal models of myocardial and peripheral vascular ischemia has prompted the translation of therapeutic angiogenesis from bench to bedside. The enthusiasm generated from early, open-label, safety trials using primarily vascular endothelial growth factors or fibroblast growth factor protein or gene therapy has been tempered by double-blind randomized placebo controlled studies that have yielded variable though potentially promising results. This article reviews the basis of angiogenic therapy, lessons learned from the latest major trials and potential refinements for future therapy.