Alessandra Pinna

Nerve Growth Factor Promotes Angiogenesis and Arteriogenesis in Ischemic Hindlimbs

Background—The neurotrophin nerve growth factor (NGF) regulates neuron survival and differentiation. Implication in neovascularization is supported by statement of NGF and its high-affinity receptor at vascular level and by NGF property of stimulating vascular endothelial cell proliferation. The present study investigated the involvement of endogenous NGF in spontaneous reparative response to ischemia. Mechanisms and therapeutic potential of NGF-induced neovascularization were examined. Methods and Results—Unilateral limb ischemia was produced in CD1 mice by femoral artery resection. By ELISA and immunohistochemistry, we documented that statement of NGF and its high-affinity receptor is upregulated in ischemic muscles. The functional relevance of this phenomenon was assessed by means of NGF-neutralizing antibody. Chronic NGF blockade abrogated the spontaneous capillarization response to ischemia and augmented myocyte apoptosis. Then we tested whether NGF administration may exert curative effects. Repeated NGF injection into ischemic adductors increased capillary and arteriole density, reduced endothelial cell and myofiber apoptosis, and accelerated perfusion recovery, without altering systemic hemodynamics. In normoperfused muscles, NFG-induced capillarization was blocked by vascular endothelial growth factor–neutralizing antibodies, dominant-negative Akt, or NO synthase inhibition. Conclusions—These results indicate that NGF plays a functional role in reparative neovascularization. Furthermore, supplementation of the growth factor promotes angiogenesis through a vascular endothelial growth factor-Akt-NO–mediated mechanism. In the setting of ischemia, potentiation of NGF pathway stimulates angiogenesis and arteriogenesis, thereby accelerating hemodynamic recovery. NGF might be envisaged as a utilitarian target for the treatment of ischemic vascular disease.

Protease-Activated Receptor-2 Stimulates Angiogenesis and Accelerates Hemodynamic Recovery in a Mouse Model of Hindlimb Ischemia

Abstract— Proteinase-activated receptors (PAR-2) are expressed by the cardiovascular system and mediate vasodilation, plasma protein extravasation, and endothelial cell proliferation, all regarded as essential steps for neovascularization. We investigated the angiogenic action of PAR-2 signaling in vivo. The effect of the PAR-2 activating peptide (PAR-2AP, SLIGRL-NH2) was assessed in the absence of ischemia, and the therapeutic potential of PAR-2AP and the PAR-2 agonist trypsin (at 300 and 1.5 nmol IM daily for 21 days, respectively) was also tested in mice subjected to unilateral limb ischemia. PAR-2AP increased capillarity in normoperfused adductor skeletal muscles, whereas neither the vehicle of the PAR2-AP nor the PAR-2 reverse peptide (PAR-2RP, LRGILS-NH2) did produce any effect. In addition, both PAR-2AP and trypsin enhanced reparative angiogenic response to limb ischemia, an effect that was not produced by PAR-2RP or the vehicle of PAR-2 agonists. Potentiation of reparative angiogenesis by PAR-2AP or trypsin resulted in an accelerated hemodynamic recovery and enhanced limb salvage. In conclusions, our study is the first to demonstrate the angiogenic potential of PAR-2 stimulation in vivo. If similar effects occur in humans, PAR-2AP agonists could have some therapeutic potential for the treatment of tissue ischemia.

Prevention of Diabetes-Induced Microangiopathy by Human Tissue Kallikrein Gene Transfer

Background—Microvascular insufficiency represents a major cause of end-organ failure among diabetics. Methods and Results—In streptozotocin-induced diabetic mice, we evaluated the potential of human tissue kallikrein (hTK) gene as a sole therapy against peripheral microangiopathy. Local delivery of hTK gene halted the progression of microvascular rarefaction in hindlimb skeletal muscle by inhibiting apoptosis, thus ensuring an improved hemodynamic recovery in case of supervening vascular occlusion. The curative action of hTK did not necessitate insulin supplementation. Application of gene therapy at a stage of established microangiopathy stimulated vascular regeneration. Conclusions—Our studies indicate that hTK may represent a useful tool for the treatment of microvascular complications in diabetics.

Angiotensin AT1 receptor signalling modulates reparative angiogenesis induced by limb ischaemia

The concept that angiotensin II exerts pro‐angiogenic activity is not universally accepted. We evaluated whether inhibition of the renin‐angiotensin system (RAS) would influence reparative angiogenesis in a murine model of limb ischaemia. Perfusion recovery following surgical removal of the left femoral artery was analysed by laser Doppler flowmetry in mice given the ACE inhibitor ramipril (1 mg kg−1 per day), the AT1 antagonist losartan (15 mg kg−1 per day), or vehicle. Muscular capillarity was examined at necroscopy. Ramipril‐induced effects were also studied under combined blockade of kinin B1 and B2 receptors. Furthermore, the effects of ischaemia on AT1 gene expression and ACE activity were determined. In untreated mice, muscular AT1a gene expression was transiently decreased early after induction of limb ischaemia, whereas AT1b mRNA was up‐regulated. ACE activity was reduced in ischaemic muscles at 1 and 3 days. Gene expression of AT1 isoforms as well as ACE activity returned to basal values by day 14. Spontaneous neovascularization allowed for complete perfusion recovery of the ischaemic limb after 21 days. Reparative angiogenesis was negatively influenced by either ramipril (P<0.02) or losartan (P<0.01), leading to delayed and impaired post‐ischaemic recovery (50 – 70% less compared with controls). Ramipril‐induced effects remained unaltered under kinin receptor blockade. The present study indicates that (a) expression of angiotensin II AT1 receptors and ACE activity are modulated by ischaemia, (b) ACE‐inhibition or AT1 antagonism impairs reparative angiogenesis, and (c) intact AT1 receptor signalling is essential for post‐ischaemic recovery. These results provide new insights into the role of the RAS in vascular biology and suggest cautionary use of ACE inhibitors and AT1 antagonists in patients at risk for developing peripheral ischaemia.

Role of calcitonin gene-related peptide and kinins in post-ischemic intestinal reperfusion

The involvement of kinins, calcitonin gene-related peptide (CGRP), and tachykinins during mesenteric post-ischemic reperfusion was studied in anesthetized rats by using antagonists for bradykinin (BK) B1, BK B2, CGRP1, or tachykinin NK1 receptor, or by capsaicin-induced desensitization. B1, B2, or CGRP1 receptor antagonists or desensitization attenuated the transient hypotension and plasma protein and leukocyte infiltration of intestinal wall observed during post-ischemic reperfusion. These effects were abolished by the combination of B2 and CGRP1 blockade as well as by B2 antagonism in capsaicinized rats, while NK1 blockade was ineffective. Our results suggest that kinins and CGRP contribute to systemic vasodilatation and microvascular leakage during mesenteric reperfusion. Pharmacological blockade of these systems could help preventing hypotension and intestinal injury consequent to reperfusion.

Ramipril improves hemodynamic recovery but not microvascular response to ischemia in spontaneously hypertensive rats.

BACKGROUND Angiotensin converting enzyme (ACE) inhibition exerts positive effects on the microvasculature of normotensive animals, although this concept is not universally accepted. Recently, ACE inhibitors have been suggested to be useful for rescue in peripheral ischemia. METHODS We investigated whether chronic treatment with the ACE inhibitor ramipril may have a positive impact on the defective healing response to ischemia that is typical of spontaneously hypertensive rats (SHR). Unilateral limb ischemia was induced in 20-week-old SHR by surgically removing the left femoral artery. Rats were allowed to regain consciousness and then were randomly allocated to treatment with ramipril (1 mg/kg body weight in drinking water) or vehicle for 28 days. RESULTS The SHR failed to develop reparative angiogenesis in response to ischemia, thus having inadequate perfusion recovery. Ramipril reduced both tail-cuff systolic blood pressure (180 +/- 7 v 207 +/- 2 mm Hg in the vehicle group at 28 days, P < .05) and intra-arterial mean blood pressure (115 +/- 6 v 135 +/- 5 mm Hg in the vehicle group, P < .05). These effects were associated with increased responsiveness to endothelium-dependent vasodilatation by acetylcholine. Treatment with ramipril did not influence muscular capillary and arteriole density but accelerated the rate of perfusion recovery, leading to complete healing within 28 days after surgery. CONCLUSIONS These results indicate that ACE inhibition by ramipril may be useful for the treatment of peripheral vascular complications in hypertension.