Paolo Madeddu

Nerve growth factor promotes reparative angiogenesis and inhibits endothelial apoptosis in cutaneous wounds of Type 1 diabetic mice

Aims/hypothesisThe neurotrophin nerve growth factor (NGF) is pro-angiogenic and facilitates wound repair. The present study was conducted to (i) assess the statement of NGF system components in diabetic wounds and (ii) evaluate whether NGF supplementation could prevent impairment of wound neoangiogenesis by diabetes.MethodsSkin wounds were produced in the interscapular region of streptozotocin-induced diabetic mice. NGF (1xa0µg per day in PBS) or vehicle was applied onto the ulcers for 3 days after punching. Non-diabetic mice were used as controls.ResultsIn wounds of untreated diabetic mice, endogenous levels of immunoreactive NGF were lower than those in wounds of non-diabetic mice (p<0.01). Immunohistochemical analysis showed down-regulation of tyrosine kinase receptor-A (TrkA) and up-regulation of p75 receptor in granulation tissue microvasculature. Local NFG administration prevented diabetes-induced expressional alterations, enhanced reparative capillarisation (p<0.01), and accelerated wound closure (p<0.01). This was associated with a three-fold increase in endothelial cell proliferation (p<0.01), while apoptosis was reduced by 50% (p<0.05). Quantitative RT-PCR documented a 5.5-fold increase in the expression of vascular endothelial growth factor-A (VEGF-A) by exogenous NGF in diabetic tissues (p<0.01). In in vitro preparations of human endothelial cells from derma, NGF increased the release of immunoreactive VEGF-A, and reduced high-glucose-induced apoptosis (p<0.05), the latter effect being inhibited by a VEGF-A receptor-2 antagonist.Conclusions/interpretationDiabetic ulcers display distinct alterations in reparative angiogenesis and in the expression of NGF and its receptors. NGF supplementation corrects endogenous liabilities, facilitates vascular regeneration, and suppresses endothelial apoptosis seemingly via VEGF-A. Our findings unravel new mechanisms responsible for NGF reparative action.

Identification of the prosurvival activity of nerve growth factor on cardiac myocytes

Neurotrophins (NTs) control neuron survival and regeneration. Recent research showed that NTs possess cardiovascular actions. In this study, we investigated the hypothesis that the NT nerve growth factor (NGF) prevents cardiomyocyte apoptosis. We demonstrated that cultured rat neonatal cardiomyocytes (RNCMs) produce NGF and express its trkA (tropomyosin-related receptor A (NGF high-affinity receptor)) receptor. RNCMs given a neutralizing antibody for NGF or the trkA inhibitor K252a underwent apoptosis, thus suggesting that NGF is an endogenous prosurvival factor for cardiomyocytes. Adenovirus (Ad)-mediated NGF overexpression protected RNCMs from apoptosis induced by either hypoxia/reoxygenation or angiotensin II (AngII). Similarly, recombinant NGF inhibited AngII-induced apoptosis in isolated rat adult cardiomyocytes. Finally, in a rat model of myocardial infarction, NGF gene transfer promoted cardiomyocyte survival. In RNCMs, recombinant NGF induced trkA phosphorylation, followed by Ser473 phosphorylation and nuclear translocation of phospho-protein kinase B (Akt). In response to Akt activation, Forkhead transcription factors Foxo-3a and Foxo-1 were phosphorylated and excluded from the nucleus. The prosurvival effect of adenoviral vector carrying the human NGF gene was inhibited in vitro by K252a, LY294002 (a pan-phosphatidyl inositol 3-kinase – PI3K – inhibitor), an Akt small interfering RNA, and adenoviruses carrying a dominant negative mutant form of Akt (Ad.DN.Akt) or an Akt-resistant Foxo-3a (Ad.AAA-Foxo-3a). These results newly demonstrate the cardiac prosurvival action of NGF and provide mechanistic information on the signaling pathway, which encompasses trkA, PI3K-Akt, and Foxo.

Nerve growth factor supplementation reverses the impairment, induced by Type 1 diabetes, of hindlimb post-ischaemic recovery in mice

Aims/hypothesisType 1 diabetes increases the risk of peripheral ischaemia and impairs recovery once ischaemia occurs, probably because the healing process is hampered by diabetes-induced endothelial dysfunction. In normoglycaemic mice subjected to limb ischaemia, blockade of nerve growth factor (NGF) compromises reparative angiogenesis. In the present study, we evaluated if expressional alterations of endogenous NGF system components are associated with diabetes-related impairment in neovascularisation. In addition, we tested whether the correction of NGF liabilities benefits post-ischaemic healing of Type 1 diabetic animals.MethodsUnilateral hindlimb ischaemia was produced in streptozotocin-induced Type 1 diabetic mice. Purified murine NGF (20xa0µg daily for 14 days) or PBS were injected into ischaemic adductors. Non-diabetic mice given PBS served as controls. Hindlimb blood flow was analysed sequentially for up to 14 days. At necroscopy, adductors were removed for quantification of microvessel density, endothelial cell apoptosis and NGF receptor expression. NGF content was determined by ELISA three days after ischaemia. In vitro, we tested whether NGF protects endothelial cells from apoptosis induced by high glucose and whether vascular endothelial growth factor-A (VEGF-A) is involved in this beneficial effect.ResultsMuscles removed from Type 1 diabetic mice showed reduced NGF content and up-regulation of the NGF p75 receptor. NGF supplementation promoted capillarisation and arteriogenesis, reduced apoptosis, and accelerated blood flow recovery. NGF stimulated VEGF-A production by human endothelial cells incubated in high-glucose medium and conferred resistance against high-glucose-induced apoptosis via a VEGF-A-mediated mechanism.Conclusions/interpretationNGF protects endothelial cells from apoptosis induced by Type 1 diabetes and facilitates reparative neovascularisation. The findings may open up new therapeutic options for the treatment of diabetic complications.

Regulation of bradykinin B2‐receptor expression by oestrogen

1 Tissue kallikrein is overexpressed in the kidney of female rats, this sexual dimorphism being associated with a greater effect of early blockade of bradykinin B2‐receptors on female blood pressure phenotype. We evaluated the effect of ovariectomy and oestradiol benzoate (50u2003μgu2003kg−1 every two days for two weeks) on the vasodepressor response to intra‐arterial injection of bradykinin (150–900u2003ngu2003kg−1) and on the expression of bradykinin B2‐receptors. 2 Ovariectomy reduced the magnitude of the vasodepressor response to bradykinin and unmasked a secondary vasopressor effect. Oestrogen replacement restored the vasodepressor response to bradykinin in ovariectomized rats. 3 The vasodepressor responses to sodium nitroprusside (3–18u2003μgu2003kg−1), acetylcholine (30–600u2003ngu2003kg−1), desArg9‐bradykinin (150–900u2003ngu2003kg−1) or prostaglandin E2 (30–600u2003ngu2003kg−1) were significantly reduced by ovariectomy. Oestrogen restored to normal the responses to desArg9‐bradykinin, acetylcholine and prostaglandin E2, but not that to sodium nitroprusside. 4 B2‐receptor mRNA levels were decreased by ovariectomy in the aorta and kidney and they were restored to normal levels by oestrogen. Neither ovariectomy nor oestradiol affected receptor expression in the heart and uterus. 5 These results indicate that oestrogen regulates B2‐receptor gene expression and function. Since kinins exert a cardiovascular protective action, reduction in their vasodilator activity after menopause might contribute to the increased risk of pathological cardiovascular events. Conversely, the cardioprotective effects of oestrogen replacement might be, at least in part, mediated by activation of the kallikrein‐kinin system.

Paracrine control of vascularization and neurogenesis by neurotrophins.

The neuronal system plays a fundamental role in the maturation of primitive embryonic vascular network by providing a paracrine template for blood vessel branching and arterial differentiation. Furthermore, postnatal vascular and neural regeneration cooperate in the healing of damaged tissue. Neurogenesis continues in adulthood although confined to specific brain regions. Following ischaemic insult, neural staminal cells contribute towards the healing process through the stimulation of neurogenesis and vasculogenesis. Evidence indicates that nerves and blood vessels exert a reciprocal control of their own growth by paracrine mechanisms. For instance, guidance factors, including vascular endothelial growth factor A (VEGF‐A) and semaphorins, which share the ability of binding neuropilin receptors, play a pivotal role in the tridimensional growth pattern of arterial vessels and nerves. Animal models and clinical studies have demonstrated a role of VEGF‐A in the pathogenesis of ischaemic and diabetic neuropathies. Further, supplementation with VEGF‐A ameliorates neuronal recovery by exerting protective effects on nerves and stimulating reparative neovascularization. Human tissue kallikrein, a recently discovered angiogenic and arteriogenic factor, accelerates neuronal recovery by stimulating the growth of vasa nervorum. Conversely, the neurotrophin nerve growth factor, known to regulate neuronal survival and differentiation, is now regarded as a stimulator of angiogenesis and arteriogenesis. These results indicate that angiogenesis and neurogenesis are paracrinally regulated by growth factors released by endothelial cells and neurons. Supplementation of these growth factors, alone or in combination, could benefit the treatment of ischaemic diseases and neuropathies.

Angiogenesis gene therapy to rescue ischaemic tissues: achievements and future directions

Ischaemic diseases are characterized by an impaired supply of blood resulting from narrowed or blocked arteries that starve tissues of needed nutrients and oxygen. Coronary‐atherosclerosis induced myocardial infarction is one of the leading causes of mortality in developed countries. Ischaemic disease also affects the lower extremities. Considerable advances in both surgical bypassing and percutaneous revascularization techniques have been reached. However, many patients cannot benefit from these therapies because of the extension of arterial occlusion and/or microcirculation impairment. Consequently, the need for alternative therapeutic strategies is compelling. An innovative approach consists of stimulating collateral vessel growth, a natural host defence response that intervenes upon occurrence of critical reduction in tissue perfusion ( Isner & Asahara, 1999 ). This review will debate the relevance of therapeutic angiogenesis for promotion of tissue repair. The following issues will receive attention: (a) vascular growth patterns, (b) delivery systems for angiogenesis gene transfer, (c) achievements of therapeutic angiogenesis in myocardial and peripheral ischaemia, and (d) future directions to improve effectiveness and safety of vascular gene therapy.

Role of nitric oxide synthase inhibition in the acute hypertensive response to intracerebroventricular cadmium

In the rat, intracerebroventricular (i.c.v.) injection of cadmium, a pollutant with long biological half‐life, causes a sustained increase in blood pressure at doses that are ineffective by peripheral route. Since cadmium inhibits calcium‐calmodulin constitutive nitric oxide (NO) synthase in cytosolic preparations of rat brain, this mechanism may be responsible for the acute pressor action of this heavy metal. To test this possibility, we evaluated the effect of i.c.v. injection of 88u2003nmol cadmium in normotensive unanaesthetized Wistar rats, which were i.c.v. pre‐treated with: (1) saline (control), (2) L‐arginine (L‐Arg), to increase the availability of substrate for NO biosynthesis, (3) D‐arginine (D‐Arg), (4) 3‐[4‐morpholinyl]‐sydnonimine‐hydrochloride (SIN‐1), an NO donor, or (5) CaCl2, a cofactor of brain calcium‐calmodulin‐dependent cNOSI. In additional experiments, the levels of L‐citrulline (the stable equimolar product derived from enzymatic cleavage of L‐Arg by NO synthase) were determined in the brain of vehicle‐ or cadmium‐treated rats. The pressor response to cadmium reached its nadir at 5u2003min (43±4u2003mmHg) and lasted over 20u2003min in controls. L‐Citrulline/protein content was reduced from 35 up to 50% in the cerebral cortex, pons, hippocampus, striatus, hypothalamus (P<0.01) of cadmium‐treated rats compared with controls. Central injection of NG nitro‐L‐arginine‐methylester (L‐NAME) also reduced the levels of L‐citrulline in the brain. Both the magnitude and duration of the response were attenuated by 1.21 and 2.42u2003μmol SIN‐1 (32±3 and 15±4u2003mmHg, P<0.05), or 1u2003μmol CaCl2 (6±4u2003mmHg, P<0.05). Selectivity of action exerted by SIN‐1 was confirmed by the use of another NO donor, S‐nitroso‐N‐acetyl‐penicillamine (SNAP). Both L‐Arg and D‐Arg caused a mild but significant attenuation in the main phase of the pressor response evoked by cadmium. However, only L‐Arg reduced the magnitude of the delayed, pressor response. Despite their similarity in ability to attenuate the cadmium‐induced pressure effect, L‐Arg and its isomer exerted differential biochemical changes in brain L‐citrulline, as L‐Arg normalized cadmium‐induced reduction in L‐citrulline levels, whereas i.c.v. D‐Arg did not. We conclude that the pressor effect of i.c.v. cadmium is due, at least in part, to reduced NO formation, consequent to inhibition of brain NO synthase. Accumulation of cadmium in the central nervous system could interfere with central mechanisms (including NO synthase) implicated in the regulation of cardiovascular function.

Targeting kinin receptors for the treatment of tissue ischaemia

Kinins, the biological end-products of the kallikrein-kininogen system, influence many aspects of the cellular function. Interest in this peptidergic system has been renewed recently by the discovery that kinins exert cardiovascular protective effects and promote post-ischaemic recovery by stimulating vascular growth. Pharmacological and genetic studies indicate that induction of kallikrein and kinin receptors by ischaemia is functionally relevant in the natural host response that permits perfusion recovery and tissue healing. Furthermore, potentiation of the generation of kinins by continuous supply of tissue kallikrein promotes reparative angiogenesis through stimulation of the release of nitric oxide and prostaglandins. Strategies that activate kinin receptors might be applicable to the treatment of occlusive vascular disease, whereas kinin receptor antagonists could represent therapeutic reagents against pathological angiogenesis in cancer and chronic inflammatory conditions.

The bradykinin B1 receptor and the central regulation of blood pressure in spontaneously hypertensive rats

We evaluated if the brain bradykinin (BK) B1 receptor is involved in the regulation of blood pressure (BP) in conscious rats. Basal mean BP and HR were 115±2 and 165±3u2003mmHg and 345±10 and 410±14 beats min−1 in Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR), respectively. Intracerebroventricular (i.c.v.) injection of 1u2003nmol B1 receptor agonist Lys‐desArg9‐BK significantly increased the BP of WKY and SHR by 7±1 and 19±2u2003mmHg, respectively. One nmol Sar[D‐Phe8]‐desArg9‐BK, a kininase‐resistant B1 agonist, increased the BP of WKY and SHR by 19±2 and 17±2u2003mmHg, respectively and reduced HR in both strains. I.c.v. injection of 0.01u2003nmol B1 antagonists, LysLeu8‐desArg9‐BK or AcLys[D‐βNal7,Ile8]‐desArg9‐BK (R715), significantly decreased mean BP in SHR (by 9±2u2003mmHg the former and 14±3u2003mmHg the latter compound), but not in WKY. In SHR, the BP response to R715 was associated to tachycardia. I.c.v. Captopril, a kininase inhibitor, increased the BP of SHR, this response being partially prevented by i.c.v. R715 and reversed into a vasodepressor effect by R715 in combination with the B2 antagonist Icatibant. I.c.v. antisense oligodeoxynucleotides (ODNs) targeted to the B1 receptor mRNA decreased BP in SHR, but not in WKY. HR was not altered in either strain. Distribution of fluorescein‐conjugated ODNs was detected in brain areas surrounding cerebral ventricles. Our results indicate that the brain B1 receptor participates in the regulation of BP. Activation of the B1 receptor by kinin metabolites could participate in the pathogenesis of hypertension in SHR.

Brain kinins are responsible for the pressor effect of intracerebroventricular captopril in spontaneously hypertensive rats.

The role of the brain kallikrein-kinin system in the regulation of arterial blood pressure of normotensive and spontaneously hypertensive rats was evaluated. Intracerebroventricular administration of the kinin antagonist [DArg0]Hyp3-Thi5,8[DPhe7]bradykinin caused no change in mean blood pressure in Wistar-Kyoto, Sprague-Dawley, or spontaneously hypertensive rats. The antagonist proved to be very potent in blocking the pressor effect of intracerebroventricular bradykinin (32 +/- 3 vs. 3 +/- 1 mm Hg, p less than 0.01). It was specific, as the pressor effect induced by other unrelated peptides was similar during the infusion of either vehicle or kinin antagonist (angiotensin II, 25 +/- 4 vs. 26 +/- 2 mm Hg; prostaglandin E2, 48 +/- 3 vs. 47 +/- 8 mm Hg; norepinephrine, 17 +/- 2 vs. 18 +/- 2 mm Hg; leucine-enkephaline, 15 +/- 2 vs. 16 +/- 1 mm Hg; neurotensin, 18 +/- 2 vs. 19 +/- 1 mm Hg; substance P, 19 +/- 2 vs. 19 +/- 2 mm Hg). Intracerebroventricular administration of 1 mg captopril, an inhibitor of kininase II (one of the enzymes responsible for kinin degradation), caused no change in mean blood pressure in normotensive rats, whereas it increased mean blood pressure by 44 +/- 9 mm Hg (p less than 0.01) in spontaneously hypertensive rats. This increase in mean blood pressure was blocked and then reversed into a hypotensive effect (22 +/- 6 mm Hg, p less than 0.05) during the infusion of kinin antagonist. Our data suggest that the pressor effect induced by intracerebroventricular captopril is due to a transient elevation in endogenous brain kinin levels, supporting the hypothesis that the brain kallikrein-kinin system plays a role in the central regulation of blood pressure in spontaneously hypertensive rats.