Maria Bonaria Salis

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.

Dilated and Failing Cardiomyopathy in Bradykinin B2 Receptor Knockout Mice

BACKGROUND The activation of B(2) receptors by kinins could exert cardioprotective effects in myocardial ischemia and heart failure. METHODS AND RESULTS To test whether the absence of bradykinin B(2) receptors may affect cardiac structure and function, we examined the developmental changes in blood pressure (BP), heart rate, and heart morphology of bradykinin B(2) receptor gene knockout (B(2)(-/-)), heterozygous (B(2)(+/-)), and wild-type (B(2)(+/+)) mice. The BP of B(2)(-/-) mice, which was still normal at 50 days of age, gradually increased, reaching a plateau at 6 months (136+/-3 versus 109+/-1 mm Hg in B(2)(+/+), P<0.01). In B(2)(+/-) mice, BP elevation was delayed. At 40 days, the heart rate was higher (P<0.01) in B(2)(-/-) and B(2)(+/-) than in B(2)(+/+) mice, whereas the left ventricular (LV) weight and chamber volume were similar among groups. Thereafter, the LV growth rate of B(2)(-/-) and B(2)(+/-) mice was accelerated, leading at 360 days to a LV weight-to-body weight ratio that was 9% and 17% higher, respectively, than that of B(2)(+/+) mice. In B(2)(-/-) mice, hypertrophy was associated with a marked chamber dilatation (42% larger than that of B(2)(+/+) mice), an elevation in LV end-diastolic pressure (25+/-3 versus 5+/-1 mm Hg in B(2)(+/+) mice, P<0.01), and reparative fibrosis. CONCLUSIONS The disruption of the bradykinin B(2) receptor leads to hypertension, LV remodeling, and functional impairment, implying that kinins are essential for the functional and structural preservation of the heart.

Local delivery of human tissue kallikrein gene accelerates spontaneous angiogenesis in mouse model of hindlimb ischemia

BackgroundHuman tissue kallikrein (HK) releases kinins from kininogen. We investigated whether adenovirus-mediated HK gene delivery is angiogenic in the context of ischemia. Methods and ResultsHindlimb ischemia, caused by femoral artery excision, increased muscular capillary density (P <0.001) and induced the expression of kinin B1 receptor gene (P <0.05). Pharmacological blockade of B1 receptors blunted ischemia-induced angiogenesis (P <0.01), whereas kinin B2 receptor antagonism was ineffective. Intramuscular delivery of adenovirus containing the HK gene (Ad.CMV-cHK) enhanced the increase in capillary density caused by ischemia (969±32 versus 541±18 capillaries/mm2 for control, P <0.001), accelerated blood flow recovery (P <0.01), and preserved energetic charge of ischemic muscle (P <0.01). Chronic blockade of kinin B1 or B2 receptors prevented HK-induced angiogenesis. ConclusionsHK gene delivery enhances the native angiogenic response to ischemia. Angiogenesis gene therapy with HK might be applicable to peripheral occlusive vascular disease.

Transplantation of low dose CD34+Kdr+ cells promotes vascular and muscular regeneration in ischemic limbs

Hematopoietic progenitor cell transplantation can contribute to revascularization of ischemic tissues. Yet, the optimal cell population to be transplanted has yet to be determined. We have compared the therapeutic potential of two subsets of human cord blood CD34+ progenitors, either expressing the VEGF‐A receptor 2 (KDR) or not. In serum‐free starvation culture, CD34+KDR+ cells reportedly showed greater resistance to apoptosis and ability to release VEGF‐A, as compared with CD34+KDR− cells. When injected into the hind muscles in immunodeficient SCIDbg mice subjected to unilateral ischemia, a low number (103) of CD34+KDR+ cells improved limb salvage and hemodynamic recovery better than a larger dosage (104) of CD34+KDR− cells. The neovascularization induced by KDR+ cells was significantly superior to that promoted by KDR− cells. Similarly, endothelial cell apoptosis and interstitial fibrosis were significantly attenuated by KDR+ cells, which differentiated into mature human endothelial cells and also apparently skeletal muscle cells. This study demonstrates that a low number of CD34+KDR+ cells favors reparative neovascularization and possibly myogenesis in limb ischemia, suggesting the potential use of this cell population in regenerative medicine.

Cardiovascular Effects of Nociceptin in Unanesthetized Mice

We evaluated the systemic hemodynamic effects induced by nociceptin (NC) and NC-related peptides, including the NC receptor antagonist [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2 ([F/G]NC(1-13)NH2) in unanesthetized normotensive Swiss Morini mice. Bolus intravenous injection of NC decreased mean blood pressure and heart rate. The hypotensive response to 10 nmol/kg NC lasted <10 minutes, whereas a more prolonged hypotension was evoked by 100 nmol/kg (from 114+/-3 to 97+/-2 mm Hg at 10 minutes, P<0.01). The latter dose reduced heart rate from 542+/-43 to 479+/-31 beats/min (P<0.05) and increased aortic blood flow by 41+/-5% (P<0.05). Hypotension and bradycardia were also evoked by NC(1-17)NH2 and NC(1-13)NH2 fragments, whereas NC(1-13)OH and NC(1-9)NH2 were ineffective. Thiorphan, an inhibitor of neutral endopeptidase 24.11, enhanced the hypotension induced by NC(1-13)NH2 and revealed the ability of NC(1-13)OH to decrease mean blood pressure. [F/G]NC(1-13)NH2, a recently synthesized antagonist of the NC receptor, did not alter basal mean blood pressure or heart rate, but it prevented the hypotension, bradycardia, and increase in aortic blood flow evoked by NC. In contrast, [F/G]NC(1-13)NH2 did not alter the hypotension induced by bradykinin or endomorphin-1 (a micro-receptor agonist), and the bradycardia induced by leu-enkephalin (a delta-receptor agonist) or U504885 (a synthetic kappa-receptor agonist). In conclusion, NC and some of its fragments cause hypotension and bradycardia and increase aortic blood flow in mice, with the NC(1-13) sequence being critical for these biological effects. Our results also demonstrate that the compound [F/G]NC(1-13)NH2 is a potent and selective antagonist of the NC receptor in vivo.

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.

Adenovirus-Mediated Human Tissue Kallikrein Gene Delivery Induces Angiogenesis in Normoperfused Skeletal Muscle

Abstract—We investigated whether local delivery of the tissue kallikrein gene induces angiogenesis in normoperfused mouse hindlimb muscles. Intramuscular injection of adenovirus containing the human tissue kallikrein gene under the control of a cytomegalovirus enhancer/promoter sequence resulted in local production and release of recombinant human tissue kallikrein, whereas transgene expression was absent in muscles of the contralateral hindlimb. Angiogenesis in infected muscles was documented by histological evidence of increased capillary density. In contrast, no angiogenic effect was seen either in the ipsilateral gastrocnemius or contralateral hindlimb muscles. Neovascularization was associated with a transient increase in muscular blood flow as determined by laser Doppler flowmetry. We also investigated the mechanisms of kallikrein-induced angiogenesis. We found that the angiogenic response to kallikrein was abolished by chronic blockade of the kinin B1 or B2 receptor or by inhibition of nitric oxide synthase. In addition, inhibition of cyclooxygenase-2 by nimesulide significantly reduced kallikrein-induced effects. These results indicate that (1) human tissue kallikrein acts as an angiogenic factor in normoperfused skeletal muscle and (2) nitric oxide and prostacyclin are essential mediators of kallikrein-induced angiogenesis. Our findings provide new insights into the role of the tissue kallikrein-kinin system in vascular biology.

Rescue of Impaired Angiogenesis in Spontaneously Hypertensive Rats by Intramuscular Human Tissue Kallikrein Gene Transfer

Angiogenesis represents a compensatory response targeted to preserve the integrity of tissues subjected to ischemia. The aim of the present study was to examine whether reparative angiogenesis is impaired in spontaneously hypertensive rats (SHR), as a function of progression of hypertension. In addition, the potential of gene therapy with human tissue kallikrein (HK) in revascularization was challenged in SHR and normotensive Wistar-Kyoto rats (WKY) that underwent excision of the left femoral artery. Expression of vascular endothelial growth factor and HK was upregulated in ischemic hindlimb of WKY but not of SHR. Capillary density was increased in ischemic adductor muscle of WKY (from 266±20 to 633±73 capillaries/mm2 at 28 days, P <0.001), whereas it remained unchanged in SHR (from 276±20 to 354±48 capillaries/mm2, P =NS), thus compromising perfusion recovery as indicated by reduced plantar blood flow ratio (0.61±0.08 versus 0.92±0.07 in WKY at 28 days, P <0.05). In separate experiments, saline or 5×109 pfu adenovirus containing the HK gene (Ad.CMV-cHK) or the &bgr;-galactosidase gene (Ad.CMV-LacZ) was injected intramuscularly at 7 days after the induction of ischemia. Ad.CMV-cHK augmented capillary density and accelerated hemodynamic recovery in both strains, but these effects were more pronounced in SHR (P <0.01). Our results indicate that native angiogenic response to ischemia is impaired in SHR, possibly as a result of defective modulation of endothelial cell mitogens. Supplementation with kallikrein, one of the growth factors found to be deficient in SHR, restores physiological angiogenic response utilitarian for tissue healing. Our discoveries may have important implications in vascular medicine for therapeutic benefit.

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.

Cardiac hypertrophy and microvascular deficit in kinin B2 receptor knockout mice.

Abstract—Experimental and clinical evidence suggests kinin involvement in adaptive myocardial growth. Kinins are growth-inhibitory to cardiomyocytes. Knockout of kinin B2 receptor (B2R) signaling causes dilated and failing cardiomyopathy in 129/J mice, and a 9-bp deletion polymorphism of human B2R is associated with reduced receptor expression and exaggerated left ventricular growth response to physical stress. We reasoned that genetic background and aging may significantly influence the impact of B2R mutation on cardiac phenotype. The theory was challenged in C57BL/6 mice, a strain that naturally differs from the 129/J strain, carrying 1 instead of 2 renin genes. C57BL/6 B2R knockouts (B2R-KO) showed higher blood pressure and heart rate levels (P <0.05) compared with wild-type controls (WT) at all ages examined. At 12 months, left ventricular contractility and diastolic function were mildly altered (P <0.05) and histological and morphological analyses revealed ventricular hypertrophy and cardiomyocyte enlargement in B2R-KO (P <0.01). Reparative fibrosis was enhanced by 208% and capillary density reduced by 38% (P <0.01). Functional and structural alterations induced by B2R deletion in C57BL/6 mice were less severe than those reported previously in the 129/J strain. We conclude that interaction of B2R signaling with other genetic determinants influences aging-related changes in myocardial structure and function. These findings may help us understand the role of kinins in the development of cardiac failure.