Claudia Rossini

Endothelin-1 as a potential marker of melatonin's therapeutic effects in smoking-induced vasculopathy.

AIMS Smoking is a significant independent risk factor for cardiovascular disease. Among the chemicals present in the cigarette smoke, nicotine is responsible for much of the damage; it induces marked vessel morphological dysfunction and vasoconstriction. Unfortunately, pharmacological or behavioural treatment is not useful against cigarette smoking. The purpose of this study is to test, in experimental conditions, the therapeutic ability of exogenous melatonin administered after smoking-induced vasculopathy and to evaluate the targets of its effects. MAIN METHODS Nicotine was orally administered for 28 days. Thereafter, the rats were orally treated with melatonin for another 28 days. Vessel damage, an important vasoconstrictor peptide (endothelin-1) and the oxidative stress markers were analysed. KEY FINDINGS Nicotine treatment induced marked endothelial damage and an obvious vasoconstriction in the aorta as evaluated by an increased endothelin-1 (ET-1) expression. These alterations were correlated with a reduction of endothelial nitric oxide synthase (eNOS) and superoxide dismutase (SOD) and with increases of heat shock protein (Hsp70) and inducible nitric oxide synthase (iNOS) activities. Melatonin not only improved the impairment of endothelial-dependent relaxation, but also induced the increase of eNOS and SOD and the reduction of iNOS and Hsp70. SIGNIFICANCE The findings indicate that nicotine is associated with an elevated synthesis of the vasoconstrictor peptide (ET-1); it also induces a reduction of nitric oxide-mediated vasodilatation (eNOS) and promotes oxidative stress in the vessel wall. We propose that melatonin should be considered as a therapeutic intervention for smokers since it reduces vasoconstriction and oxidative stress and improves endothelial physiology.

Effect of antihypertensive treatment on microvascular structure, central blood pressure and oxidative stress in patients with mild essential hypertension.

Background: It has been previously demonstrated that dihydropyridine calcium channel blockers may possess antioxidant properties and might improve vascular structure. Combination treatment with an angiotensin-converting enzyme inhibitor may have additional advantages, compared with a thiazide diuretic, in this regard. The aim of the present study was, therefore, to investigate the effects of a short-term treatment with lercanidipine, and to compare two combination treatments: lercanidipine + enalapril vs. lercanidipine + hydrochlorothiazide on structural alterations in retinal arterioles, on skin capillary density and on large artery distensibility. Patients and methods: Twenty essential hypertensive patients were included in the study and treated for 4 weeks with lercanidipine 20 mg per day orally. Then they were treated for 6 months with lercanidipine + enalapril (n = 10) or lercanidipine + hydrochlorothiazide (n = 10) combinations. Investigations were performed in basal condition, after appropriate washout of previous treatments, after 4 weeks of lercanidipine monotherapy treatment, and at the end of the combination treatment. Non-invasive measurements of wall-to-lumen ratio (W/L) and other morphological parameters of retinal arterioles using scanning laser Doppler flowmetry were performed (Heidelberg Retina Flowmeter, Heidelberg Engineering). Capillary density was evaluated by capillaroscopy, whereas pulse wave velocity and central blood pressure were assessed by the Sphygmo-Cor device (AtCor Medical West Ryde, Australia). Results: A significant improvement of W/L and of other indices of retinal artery structure was observed after treatment with lercanidipine alone, with a further improvement after treatment with lercanidipine + enalapril, whereas after treatment with lercanidipine + hydrochlorothiazide the improvement was no longer observed. A similar behaviour was observed for central SBP and DBP. Capillary density was increased only after treatment with lercanidipine + enalapril. Conclusion: Lercanidipine both in monotherapy and in combination with enalapril, was able to improve microvascular structure and to decrease central blood pressure, being thus a useful approach for both reducing blood pressure and improving vascular alterations in hypertension.

Anticontractile activity of perivascular fat in obese mice and the effect of long-term treatment with melatonin.

Aims: It has been demonstrated previously that inflammation in perivascular adipose tissue (PVAT) may be implicated in vascular dysfunction. The aim of this study was to investigate the functional responses of small mesenteric arteries in a hyperphagic animal model of obesity after chronic treatment with melatonin, an endogenous hormone with antioxidant and vasculoprotective properties. Methods and results: Ten obese mice (ob/ob) and 10 control lean mice (CLM) were treated with melatonin 100 mg/kg per day in the drinking water for 8 weeks. Mesenteric small resistance arteries were dissected and mounted on a wire myograph and a concentration-response to norepinephrine was evaluated in vessels with intact PVAT and after PVAT was removed and in the presence of iberiotoxin, a selective blocker of BKCA channels as well as under conditions of induced hypoxia in vitro. The presence of PVAT reduced the contractile response to norepinephrine in both ob/ob and CLM; however, the effect was significantly reduced in ob/ob. The anticontractile effect of PVAT completely disappeared with iberiotoxin preincubation. After melatonin treatment, inflammation was significantly ameliorated, and the contractile response in ob/ob and CLM was significantly reduced when PVAT was removed. Anticontractile effect of PVAT that is lost in obesity can be rescued using melatonin. A reduced expression of adiponectin and adiponectin receptor was observed in perivascular fat of ob/ob, whereas significant increase was observed in ob/ob treated with melatonin. Conclusion: Melatonin seems to exert a protective effect on arteries from both ob/ob and CLM, counteracting the adverse effect of hypoxia and iberiotoxin.

How to Assess Microvascular Structure in Humans

Structural alterations of subcutaneous small resistance arteries, as indicated by an increased media to lumen ratio, are frequently present in hypertensive and/or diabetic patients. However, the evaluation of microvascular structure is not an easy task. Among the methods that may be applied to humans, plethysmographic evaluation of small arteries and wire or pressure micromyography were extensively used in the last decades. Media to lumen ratio of small arteries evaluated by micromyography was demonstrated to possess a strong prognostic significance; however, its extensive evaluation is limited by the invasiveness of the assessment, since a biopsy of subcutaneous fat is needed. Non-invasive approaches were then proposed, including capillaroscopy, which provides information about microvascular rarefaction. Recently, the interest of investigators has focused on the retinal microvascular bed. In particular, a non-invasive measurement of wall thickness to internal lumen ratio of retinal arterioles using scanning laser Doppler flowmetry has been recently introduced. Preliminary data suggest a fairly good agreement between this approach and micromyographic measurements, generally considered the gold standard approach. Therefore, the evaluation of microvascular structure is progressively moving from bench to bedside, and it could represent, in the immediate future, an evaluation to be performed in all hypertensive patients, in order to obtain a better stratification of cardiovascular risk.

Circulating endothelial progenitor cells, microvascular density and fibrosis in obesity before and after bariatric surgery.

Abstract It is not known whether, in obesity, the capillary density or the number of circulating endothelial progenitor cells (EPCs) are reduced, or whether fibrosis of small vessels is also present. In addition, possible effects of weight reduction on these parameters have never been evaluated. Therefore, we investigated EPCs and capillary density in 25 patients with severe obesity, all submitted to bariatric surgery, and in 18 normotensive lean subjects and 12 hypertensive lean patients as controls. All patients underwent a biopsy of subcutaneous fat during bariatric surgery. In five patients, a second biopsy was obtained after consistent weight loss, about 1 year later, during a surgical intervention for abdominoplasty. EPCs and capillary density were reduced in obesity, and EPCs were significantly increased after weight reduction. Vascular collagen content was clearly increased in obese patients. No significant difference in vascular collagen was observed between normotensive obese patients and hypertensive obese patients. After pronounced weight reduction, collagen content was nearly normalized. No difference in stress–strain relation was observed among groups or before and after weight loss. In conclusion, our data suggest that microvascular rarefaction occurs in obesity. EPCs were significantly reduced in obese patients. Pronounced weight loss induced by bariatric surgery seems to induce a significant improvement of EPC number, but not of capillary rarefaction. A pronounced fibrosis of subcutaneous small resistance arteries is present in obese patients, regardless of the presence of increased blood pressure values. Consistent weight loss induced by bariatric surgery may induce an almost complete regression of microvascular fibrosis.

Immune Mechanisms in Hypertension

Low grade inflammation may have a key role in the pathogenesis of hypertension and cardiovascular disease. Several studies showed that both innate and adaptive immune systems may be involved, being T cells the most important players. Particularly, the balance between Th1 effector lymphocytes and Treg lymphocytes may be crucial for blood pressure elevation and related organ damage development. In the presence of a mild elevation of blood pressure, neo-antigens are produced. Activated Th1 cells may then contribute to the persistent elevation of blood pressure by affecting vasculature, kidney and perivascular fat. On the other hand, Tregs represent a lymphocyte subpopulation with an anti-inflammatory role, being their activity crucial for the maintenance of cardiovascular homeostasis. Indeed, Tregs were demonstrated to be able to protect from blood pressure elevation and from the development of organ damage, including micro and macrovascular alterations, in different animal models of genetic or experimental hypertension. In the vasculature, inflammation leads to vascular remodeling through cytokine activity, smooth muscle cell proliferation and oxidative stress. It is also known that a consistent part of ischemia-reperfusion-induced acute kidney injury is mediated by inflammatory infiltration and that Treg cell infusion have a protective role. Also the central nervous system has an important role in the maintenance of cardiovascular homeostasis. In conclusion, hypertension development involves chronic inflammatory process. Knowledge of cellular and molecular players in the progression of hypertension has dramatically improved in the last decade, by assessing the central role of innate and adaptive immunity cells and proinflammatory cytokines driving the development of target organ damage. The new concept of role of immunity, especially implicating T lymphocytes, will eventually allow discovery of new therapeutic targets that may improve outcomes in hypertension and cardiovascular or renal disease in humans and uncover an entirely novel approach in the treatment of hypertension and vascular disease.

Effect of long-term treatment with melatonin on vascular markers of oxidative stress/inflammation and on the anticontractile activity of perivascular fat in aging mice.

Some reports have suggested that inflammation in perivascular adipose tissue (PVAT) may be implicated in vascular dysfunction by causing the disappearance of an anticontractile effect. The aim of this study was to investigate the effects of chronic melatonin treatment on the functional responses of the small mesenteric arteries and on the expression of markers of inflammation/oxidative stress in the aortas of senescence-accelerated prone mice (SAMP8), a model of age-related vascular dysfunction. We investigated seven SAMP8 and seven control senescence-accelerated resistant mice (SAMR1) treated for 10 months with melatonin, as well as equal numbers of age-matched untreated SAMP8 and SAMR1. The mesenteric small resistance arteries were dissected and mounted on a wire myograph, and the concentration–response to norepinephrine was evaluated in vessels with intact PVAT and after the removal of the PVAT. The expression of markers of oxidative stress, inflammation and aging in the aortas was evaluated by immunostaining. In addition, the adiponectin content and the expression of adiponectin receptor 1 were evaluated in the visceral adipose tissue. In untreated SAMP8 mice, we observed an overexpression of oxidative stress and inflammatory markers in the vasculature compared with the controls. No anticontractile effect of the PVAT was observed in untreated SAMP8 mice. Long-term treatment of SAMP8 mice with melatonin increased the expression of some markers of vasoprotection, decreased oxidative stress and inflammation and restored the anticontractile effect of the PVAT. Decreased expression of adiponectin and adiponectin receptor 1 was also observed in visceral fat of untreated SAMP8, whereas a significant increase was observed after melatonin treatment.

Nicotine-induced morphological changes in rat aorta: the protective role of melatonin.

We analyzed the morphological changes in rat aortas during nicotine administration in order to investigate the involvement of vascular smooth muscle cells (VSMCs) in the regulation of vascular wall homeostasis. We also considered the possibility of restoring VSMC changes using melatonin as an antioxidant. We studied 4 groups of animals over 56 days. Three groups of rats were used as controls (the first without treatment, the second with melatonin alone and the third with nicotine alone). The last group of rats was orally treated with nicotine for the first 28 days and with melatonin for the last 28 days. Morphological changes in vessels were evaluated by histological procedures and immunohistochemical analysis using thrombospondin-1 (TSP-1), transforming growth factor-β1 (TGF-β1), plasminogen activator inhibitor-1 (PAI-1) and CD31 antibodies. We demonstrated that TSP-1, TGF-β1 and PAI-1 increased after nicotine administration. We believe that TSP-1 is responsible for neointima formation and that it is able to influence TGF-β1 and PAI-1 expression. Histological and immunohistochemical analysis by CD31 antibody showed that only a few endothelial cells were present in the aorta after nicotine administration compared to controls and rats treated with melatonin after nicotine administration. Moreover, histological analysis showed that neointima formation was present after nicotine treatment. Furthermore, melatonin inhibited neointima formation increasing TSP-1 expression. The ability of melatonin to inhibit neointima formation suggests that it could be a useful treatment for homeostasis of vascular walls.

Effects of a Long-Term Treatment With Aliskiren or Ramipril on Structural Alterations of Subcutaneous Small-Resistance Arteries of Diabetic Hypertensive Patients

Structural alterations of subcutaneous small-resistance arteries are associated with a worse clinical prognosis in hypertension and non–insulin-dependent diabetes mellitus. The effects of the direct renin inhibitor aliskiren on microvascular structure were never previously evaluated. Therefore, we investigated the effects of aliskiren in comparison with those of an extensively used angiotensin-converting enzyme inhibitor, ramipril, on peripheral subcutaneous small-resistance artery morphology, retinal arteriolar structure, and capillary density in a population of patients with non–insulin-dependent diabetes mellitus. Sixteen patients with mild essential hypertension and with a previous diagnosis of non–insulin-dependent diabetes mellitus were included in the study. Patients were then randomized to 1 of the 2 active treatments (aliskiren 150 mg once daily, n=9; or ramipril 5 mg once daily, n=7). Each patient underwent a biopsy of the subcutaneous fat from the gluteal region, an evaluation of retinal artery morphology (scanning laser Doppler flowmetry), and capillary density (capillaroscopy), at baseline and after 1 year of treatment. Subcutaneous small arteries were dissected and mounted on a pressurized micromyograph, and the media-to-lumen ratio was evaluated. A similar office blood pressure–lowering effect and a similar reduction of the wall-to-lumen ratio of retinal arterioles were observed with the 2 drugs. Aliskiren significantly reduced media-to-lumen ratio of subcutaneous small-resistance arteries, whereas ramipril-induced reduction of media to lumen ratio was not statistically significant. No relevant effect on capillary density was observed. In conclusion, treatment with aliskiren or ramipril was associated with a correction of microvascular structural alterations in patients with non–insulin-dependent diabetes mellitus.

Relationship Between Different Subpopulations of Circulating CD4+ T-lymphocytes and Microvascular Structural Alterations in Humans

BACKGROUND Different components of the immune system, including innate and adaptive immunity (T-effector lymphocytes and T-regulatory lymphocytes—TREGs) may be involved in the development of hypertension. In addition, it was demonstrated in animal models that TREGs may prevent angiotensin II-induced hypertension and vascular injury/inflammation. However, no data are presently available in humans about possible relationships between T-lymphocyte subtypes and microvascular structural alterations. METHODS For this purpose, in the present study, we enrolled 24 normotensive subjects and 12 hypertensive patients undergoing an elective surgical intervention. No sign of local or systemic inflammation was present. All patients underwent a biopsy of subcutaneous fat during surgery. Subcutaneous small resistance arteries were dissected and mounted on a wire myograph and the media to lumen ratio (M/L) was calculated. In addition, retinal arteriolar structure was evaluated noninvasively by scanning laser Doppler flowmetry. Capillary density in the nailfold, dorsum of the finger, and forearm were evaluated by videomicroscopy. A peripheral blood sample was obtained before surgery for assessment of T-lymphocyte subpopulations by flow cytometry. RESULTS Significant negative correlations were observed between indices of microvascular structure (M/L of subcutaneous small arteries and wall to lumen ratio of retinal arterioles) and circulating TREG lymphocytes. A direct correlation was observed between M/L of subcutaneous small arteries and circulating Th17 lymphocytes. In addition, total capillary density was correlated with a TREG effector memory subpopulation. CONCLUSION Our data suggest that some lymphocyte subpopulations may be related to microvascular remodeling, confirming previous animal data, and opening therapeutic possibilities.