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Aims Lymphatics are essential for cardiac health, and insufficient lymphatic expansion (lymphangiogenesis) contributes to development of heart failure (HF) after myocardial infarction. However, the regulation and impact of lymphangiogenesis in non-ischaemic cardiomyopathy following pressure-overload remains to be determined. Here, we investigated cardiac lymphangiogenesis following transversal aortic constriction (TAC) in C57Bl/6 and Balb/c mice, and in end-stage HF patients. Methods and results Cardiac function was evaluated by echocardiography, and cardiac hypertrophy, lymphatics, inflammation, oedema, and fibrosis by immunohistochemistry, flow cytometry, microgravimetry, and gene expression analysis. Treatment with neutralizing anti-VEGFR3 antibodies was applied to inhibit cardiac lymphangiogenesis in mice. We found that VEGFR3-signalling was essential to prevent cardiac lymphatic rarefaction after TAC in C57Bl/6 mice. While anti-VEGFR3-induced lymphatic rarefaction did not significantly aggravate myocardial oedema post-TAC, cardiac immune cell levels were increased, notably myeloid cells at 3 weeks and T lymphocytes at 8 weeks. Moreover, whereas inhibition of lymphangiogenesis did not aggravate interstitial fibrosis, it increased perivascular fibrosis and accelerated development of left ventricular (LV) dilation and dysfunction. In clinical HF samples, cardiac lymphatic density tended to increase, although lymphatic sizes decreased, notably in patients with dilated cardiomyopathy. Similarly, comparing C57Bl/6 and Balb/c mice, lymphatic remodelling post-TAC was linked to LV dilation rather than to hypertrophy. The striking lymphangiogenesis in Balb/c was associated with reduced cardiac levels of macrophages, B cells, and perivascular fibrosis at 8 weeks post-TAC, as compared with C57Bl/6 mice that displayed weak lymphangiogenesis. Surprisingly, however, it did not suffice to resolve myocardial oedema, nor prevent HF development. Conclusions We demonstrate for the first time that endogenous lymphangiogenesis limits TAC-induced cardiac inflammation and perivascular fibrosis, delaying HF development in C57Bl/6 but not in Balb/c mice. While the functional impact of lymphatic remodelling remains to be determined in HF patients, our findings suggest that under settings of pressure-overload poor cardiac lymphangiogenesis may accelerate HF development.

Aims Von Willebrand factor (VWF) is a plasma glycoprotein involved in primary hemostasis, while also having additional roles beyond hemostasis namely in cancer, inflammation, angiogenesis and potentially in vascular smooth muscle cell (VSMC) proliferation. Here, we addressed how VWF modulates VSMC proliferation and investigated the underlying molecular pathways and the in vivo pathophysiological relevance. Methods and results VWF induced proliferation of human aortic VSMCs and also promoted VSMC migration. Treatment of cells with a siRNA against αv integrin or the RGT-peptide blocking αvβ3 signaling abolished proliferation. However, VWF did not bind to αvβ3 on VSMCs through its RGD-motif. Rather, we identified the VWF A2 domain as the region mediating binding to the cells. We hypothesized the involvement of a member of the LDL-related receptor protein (LRP) family due to their known ability to act as co-receptors. Using the universal LRP-inhibitor receptor-associated protein, we confirmed LRP-mediated VSMC proliferation. siRNA experiments and confocal fluorescence microscopy identified LRP4 as the VWF-counterreceptor on VSMCs. Also co-localization between αvβ3 and LRP4 was observed via proximity ligation analysis and immuno-precipitation experiments. The pathophysiological relevance of our data was supported by VWF-deficient mice having significant reduced, if any, hyperplasia in carotid artery ligation and artery femoral denudation models. In wild-type mice, infiltration of VWF in intimal regions enriched in proliferating VSMCs was found. Interestingly, also analysis of human atherosclerotic lesions showed abundant VWF accumulation in VSMC-proliferating rich intimal areas. Conclusions VWF mediates VSMC proliferation through a mechanism involving A2 domain binding to the LRP4 receptor and integrin αvβ3 signaling. Our findings provide new insights into the mechanisms that drive physiological repair and pathological hyperplasia of the arterial vessel wall. In addition, the VWF/LRP4-axis may represent a novel therapeutic target to modulate VSMC proliferation. Translational perspective The mechanisms that drive physiological repair and pathological hyperplasia of the arterial vessel wall are complex and only partially understood. Specifically, the role of subendothelial-matrix proteins remains unclear. Here, we show that the hemostatic protein von Willebrand factor (VWF) accumulates in the vascular wall of atherosclerotic lesions and localizes to areas of vascular smooth muscle cell (VSMC) proliferation. VWF was found to use its A2-domain for binding to the VSMC-receptor LRP4, which in turn triggered outside-in signaling via integrin αVβ3, thereby inducing VSMC proliferation. Interfering with A2-domain/LRP4 interactions might offer innovative and additional therapeutical approaches to limit pathological hyperplasia.

Aims Inflammatory mediators, including blood cells and their products, contribute critically to atherogenesis, but the igniting triggers of inflammation remain elusive. Atherosclerosis develops at sites of flow perturbation, where the enhanced haemodynamic stress could initiate the atherogenic inflammatory process due to the occurrence of mechanic injury. We investigated the role of haemodynamic stress-induced breaches, allowing the entry of blood cells in the arterial intima, in triggering inflammation-driven atherogenesis. Methods and results Human coronary samples isolated from explanted hearts, (n = 47) displayed signs of blood entry (detected by the presence of iron, ferritin, and glycophorin A) in the subintimal space (54%) as assessed by histology, immunofluorescence, high resolution episcopic microscopy, and scanning electron microscopy. Computational flow dynamic analysis showed that intimal haemorrhagic events occurred at sites of flow disturbance. Experimental carotid arteries from Apoe deficient mice showed discrete endothelial breaches and intimal haemorrhagic events specifically occurring at the site of flow perturbation, within 3 days after the exacerbation of the local haemodynamic stress. Endothelial tearing was associated with increased VCAM-1 expression and, within 7 days, substantial Ly6G+ leucocytes accumulated at the sites of erythrocyte-derived iron and lipids droplets accumulation, pathological intimal thickening and positive oil red O staining. The formation of fatty streaks at the sites of intimal breaches was prevented by the depletion of Ly6G+ leucocytes, suggesting that the local injury driven by haemodynamic stress-induced breaches triggers atherogenic inflammation. Conclusion Haemodynamic-driven breaches of the arterial intima drive atherogenic inflammation by triggering the recruitment of leucocyte at sites of disturbed arterial flow.

Aims: Both leukotrienes and neutrophils have been linked to plaque destabilization. Despite being evoked, the role of leukotriene B4 (LTB4) in neutrophil recruitment to plaques and the concomitant effects of these two actors on plaque stability remain to be proven. Since both actors are elicited during endotoxaemia, a condition associated with the risk of cardiovascular events, we investigated whether endotoxaemia promotes LTB4-mediated neutrophil infiltration in plaques and explored the roles of LTB4 and neutrophils in plaque destabilization. Methods and results: Endotoxaemia induced by repeated peritoneal endotoxin injections at a non-lethal dose (1.5 mg/kg, 5 days) in chow-fed aged Apoe-/- mice (over 45 weeks old) resulted in neutrophil infiltration and activation in plaques. Subsequently to neutrophil invasion, plaques exhibited increased features of vulnerability: reduced collagen content, expanded necrotic cores, and thinned fibrous caps. These plaque features were reproduced by direct deposition of isolated neutrophils onto murine atheromatous carotid arteries in an in vivo assay. In endotoxemic mice, plaques produced increased amounts of LTB4. Genomic or pharmacological impairments of this production reduced neutrophil infiltration, collagenolysis, and apoptosis of smooth muscle cells in plaques of endotoxemic mice. Furthermore, conditioned media of human culprit plaques (CPs) contained more LTB4 than non-CPs and levels of LTB4 correlated to both neutrophil activation markers and endotoxin releases in CPs. Conclusion: These results show that the increased neutrophil recruitment elicited by LTB4 contributes to increase features of plaque destabilization in endotoxemic contexts and point out LTB4 as a potential therapeutic target in atherosclerosis.

Vascular smooth muscle cells (vSMCs) play a crucial role in both the pathogenesis of Aneurysms and Dissections of the ascending thoracic aorta (TAAD) in humans and in the associated adaptive compensatory responses, since thrombosis and inflammatory processes are absent in the majority of cases. Aneurysms and dissections share numerous characteristics, including aetiologies and histopathological alterations: vSMC disappearance, medial areas of mucoid degeneration, and extracellular matrix (ECM) breakdown. Three aetiologies predominate in TAAD in humans: (i) genetic causes in heritable familial forms, (ii) an association with bicuspid aortic valves, and (iii) a sporadic degenerative form linked to the aortic aging process. Genetic forms include mutations in vSMC genes encoding for molecules of the ECM or the TGF-β pathways, or participating in vSMC tone. On the other hand, aneurysms and dissections, whatever their aetiologies, are characterized by an increase in wall permeability leading to transmural advection of plasma proteins which could interact with vSMCs and ECM components. In this context, blood-borne plasminogen appears to play an important role, because its outward convection through the wall is increased in TAAD, and it could be converted to active plasmin at the vSMC membrane. Active plasmin can induce vSMC disappearance, proteolysis of adhesive proteins, activation of MMPs and release of TGF-β from its ECM storage sites. Conversely, vSMCs could respond to aneurysmal biomechanical and proteolytic injury by an epigenetic phenotypic switch, including constitutional overexpression and nuclear translocation of Smad2 and an increase in antiprotease and ECM protein synthesis. In contrast, such an epigenetic phenomenon is not observed in dissections. In this context, dysfunction of proteins involved in vSMC tone are interesting to study, particularly in interaction with plasma protein transport through the wall and TGF-β activation, to establish the relationship between these dysfunctions and ECM proteolysis.

Aims Despite adequate medical management, dissection of the descending aorta (type B) may develop complications, including aneurysmal progression and eventually rupture. Partial false lumen thrombosis has been identified as a marker of adverse evolution in chronic dissection. The aim of this study was to test the ability of complementary information, provided by 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) and peripheral biomarkers, to add pathophysiological significance and a prognostic value to morphological data. Methods and results We explored serial aortic 18F-FDG uptake by PET/CT imaging and plasma biomarkers in a series of 23 patients with type B dissection to predict complications from initial data and to investigate potential associations with aneurysmal expansion during follow-up. Complications occurred in 17 patients. Acute initial characteristics associated with complications were male gender ( P = 0.021), arterial hypertension ( P = 0.040), aortic dissection diameter ( P = 0.0086), partial thrombosis of the false channel ( P = 0.0046), and enhanced focal 18F-FDG uptake ( P = 0.045). During follow-up (mean 16.7 ± 8.0 months), aneurysmal expansion was associated with false lumen morphology ( P

Aims Human thoracic aneurysm of the ascending aorta (TAA) is a chronic disease characterized by dilatation of the aortic wall, which can progress to vessel dissection and rupture. TAA has several aetiologies, but all forms present common features, including tissue remodelling. Here, we determined and characterized the angiogenic process associated with TAA and its relation with wall remodelling. Methods and results Immunostaining for blood vessels showed an increased density of microvessels originating from the adventitia in the external medial layer of TAA compared with healthy aortas. Proteomic array analysis of 55 angiogenic factors in medial and adventitial layers showed different expression profiles in both tissue compartments between aneurysmal and healthy aortas. Quantification by ELISA confirmed that all forms of TAA contained higher levels of several pro- and anti-angiogenic factors, including angiopoietin-1 and -2, fibroblast growth factor-acidic, and thrombospondin-1, than that of healthy aortas. However, all groups showed comparable levels of vascular endothelial growth factor-A. Quantitative RT-PCR demonstrated that angiopoietins were overexpressed in TAA media. Immunostaining and electron microscopy revealed that neovessels had defective endothelial junctions and poor mural cell coverage. This incomplete structure was associated with the accumulation of plasminogen and albumin in the media of TAA. Conclusion We describe, for the first time, leaky neovessel formation in TAA media in association with an imbalance of angiogenic factor levels. Although the initiating mechanisms of neo-angiogenesis in TAA and the potential aetiology-related differences remain to be determined, our results suggest that neo-angiogenesis could participate in TAA wall remodelling and weakening through deposition of blood-borne zymogens.

Aims Abdominal aortic aneurysm (AAA) is a particular form of atherothrombotic disease characterized by the dilation of the aortic wall and the presence of an intraluminal thrombus (ILT). The objective of the present study was to evaluate the pro-oxidant properties of the ILT and to characterize the anti-oxidant capacity of high-density lipoproteins (HDLs). Methods and results Our results show that ILT, adventitia, and plasma from AAA patients contained high concentrations of lipid and protein oxidation products. Mediators produced within or released by the thrombus and the adventitia were shown to induce reactive oxygen species (ROS) production by cultured aortic smooth muscle cells (AoSMCs) and to trigger the onset of apoptosis (an increase in mitochondrial membrane potential). Iron chelation limited these effects. Both concentration and functionality of HDLs were altered in AAA patients. Plasma levels of Apo A-I were lower, and small HDL subclasses were decreased in AAA patients. Circulating HDLs in AAA patients displayed an impaired capacity to inhibit copper-induced low-density lipoprotein oxidation and AoSMC ROS production. Western blot analyses of HDLs demonstrated that myeloperoxidase is associated with HDL particles in AAA patients. Conclusion ILT and adventitia are a source of pro-oxidant products, in particular haemoglobin, which may impact on the wall stability/rupture in AAA. In addition, HDLs from AAA patients exhibit an impaired anti-oxidant activity. In this context, restoring HDL functionality may represent a new therapeutic option in AAA.

Vascular smooth muscle cells (VSMCs) are the stromal cells of the vascular wall, continually exposed to mechanical signals and biochemical components generated in the blood compartment. They are involved in all the physiological functions and the pathological changes taking place in the vascular wall. Owing to their contractile tonus, VSMCs of resistance vessels participate in the regulation of blood pressure and also in hypertension. VSMCs of conduit arteries respond to hypertension-induced increases in wall stress by an increase in cell protein synthesis (hypertrophy) and extracellular matrix secretion. These responses are mediated by complex signalling pathways, mainly involving RhoA and extracellular signal-regulated kinase1/2. Serum response factor and miRNA expression represent main mechanisms controlling the pattern of gene expression. Ageing also induces VSMC phenotypic modulation that could have influence on cell senescence and loss of plasticity and reprogramming. In the early stages of human atheroma, VSMCs support the lipid overload. Endocytosis/phagocytosis of modified low-density lipoproteins, free cholesterol, microvesicles, and apoptotic cells by VSMCs plays a major role in the progression of atheroma. Migration and proliferation of VSMCs in the intima also participate in plaque progression. The medial VSMC is the organizer of the inwardly directed angiogenic response arising from the adventitia by overexpressing vascular endothelial growth factor in response to lipid-stimulated peroxisome proliferator-activated receptor-γ, and probably also the organizer of the adventitial immune response by secreting chemokines. VSMCs are also involved in the response to proteolytic injury via their ability to activate blood-borne proteases, to secrete antiproteases, and to clear protease/antiprotease complexes.

Atherosclerotic plaque rupture triggers the onset of cardiovascular complications such as myocardial infarction and stroke, which represent the main cause of death in western countries. Atherogenesis is a complex process characterized by lipid retention, proteolytic injury and a chronic inflammatory response. The resulting pathological vascular remodeling involves inflammatory cell recruitment, fibrosis, smooth muscle cell proliferation, neovascularization and intraplaque hemorrhage. However, the cellular and molecular mechanisms underlying cardiovascular dysfunction remain widely unknown. The development of differential proteomics allows the identification of novel proteins whose association with the genesis of atherosclerotic plaques is at present unforeseen in the light of available data. Moreover, different strategies have been used to discover new potential biomarkers which could be related to cardiovascular risk. The multi-factorial nature of cardiovascular diseases necessitates the use of biomarkers for early detection, for monitoring the response to therapy and to predict clinical outcome. In this review, we summarize the different proteomic approaches and recent findings that will help us to understand the mechanisms implicated in the pathogenesis of atherothrombosis.

Aims Aortic valve diseases are characterized by pathological remodelling of valvular tissue but the cellular and molecular effectors involved in these processes are not well known. The role of matrix metalloproteinase (MMP)-2, MMP-9, MMP-3, MMP-7, and tissue inhibitor of matrix metalloproteinase (TIMP)-1 and TIMP-2 are investigated here. Methods and results Histological analysis of pathological valves [aortic stenosis (AS) ( n =49), aortic regurgitation (AR) ( n =23)] and control valves ( n =8) was performed. The main tissue abnormalities (calcification, inflammatory cells, and capillaries) observed in AS were less severe or absent in AR. However, both groups of pathological valves displayed similar histological signs of extracellular matrix (ECM) remodelling. Biochemical analysis of MMPs and TIMPs (gelatin and casein zymography and ELISA) was performed on valve extracts. MMP-2 activity was not significantly different in control and pathological valves. Increases in MMP-9 and MMP-3 in AS demonstrated an inflammatory state. Finally, there was a four- to seven-fold increase of TIMP-1 in pathological valves. TIMP-1, TIMP-2, and MMP-2 were synthesized by the valvular interstitial cells in primary culture. Conclusion This study demonstrates the involvement of the MMP/TIMP system in ECM remodelling of both AS and AR. These findings provide evidence of inflammatory injury more severe in AS than in AR and involvement of mesenchymal cell response.

Objective: Lower limb perfusion following arterial occlusion depends on the recruitment of collateral vessels. Blood flow through these collateral vessels may be jeopardized by hypersensitivity to vasoconstriction by serotonin (5-HT), as has been reported during hyperlipidemia and diabetes. Therefore the aim of this study was to determine the benefits of chronic treatment with SL65.0472, a mixed 5-HT1B/5-HT2A receptor antagonist, on lower limb ischemia in fatty fa / fa Zucker rats, a strain characterized by obesity, diabetes and hyperlipidemia. Comparison was made with lean control fa /+ Zucker rats. Methods: SL65.0472 (3 mg/kg/day, n =16) or its vehicle ( n =20) were administered orally for 13 days to male fatty fa / fa Zucker rats submitted to lower limb ischemia. Hindlimb ischemia was induced unilaterally by resection of the left femoral and external iliac arteries and embolization of the left internal iliac artery with microspheres. Distal perfusion was measured under mild anesthesia by laser Doppler imaging of both feet. The perfusion deficit (Δ%) was calculated before and 3, 7 and 14 days after induction of hindlimb ischemia. Twenty-four hours after the last administration of SL65.0472, muscular partial oxygen pressure, iliac blood flows, maximal vasodilatory reserve and the vasoreactivity to 5-HT were measured in both limbs. Results: Chronic administration of SL65.0472 improved the distal perfusion from day 3. At day 14, the deficit of perfusion was limited to −36±7% in SL65.0472-treated animals vs. −70±6% in the vehicle-treated group ( P

International audience; Objectives: Endothelin-1 (ET-1), plays an important role in the pathophysiology of CHF and the pulmonary endothelium is an early hemodynamic target in diastolic left ventricular dysfunction. Therefore we hypothesized that the lung is a main source of humoral endothelin in CHF and that its secretion is proportional to the degree of heart failure. Methods and results: We used rats with coronary artery ligation as an experimental model of either compensated or decompensated heart failure, depending on infarct size. Reverse transcriptase polymerase chain reaction (RT-PCR) revealed that in the lung, the expression of preproET-1 mRNA was higher in decompensated HF than in control and compensated HF rats (P

Objective: Hemodynamic stresses are considered to be important regulators of gene expression in vascular cells. In this study, we have investigated the role of shear stress on ACE expression in cultured rat vascular cells, and focused on the regulation of ACE expression in smooth muscle cells. Methods: Rat aortic endothelial cells, smooth muscle cells and fibroblasts isolated from Wistar rats were submitted to shear stress using a laminar shear flow parallel chamber. Results: A 10 dynes/cm2 shear rate for 24 h increased ACE activity in the three vascular cell types (× 2.14 in endothelial cells, × 2.9 in smooth muscle cells, × 3.33 in fibroblasts). This induction was blocked by a 24 h pre-incubation with a translation blocker (10−4M cycloheximide) showing the role of protein neosynthesis. Therefore the study was focused on smooth muscle cells and we demonstrated that the increase in ACE activity was due to an elevation in ACE mRNA level in response to a 10 dynes/cm2 shear stress for 24 h. This induction was dependent on the shear intensity ( P

Objective: To address the effect of longstanding left ventricular (LV) hypertrophy and failure on LV adenylyl cyclase (AC) gene expression, mRNA concentrations of the main cardiac AC isoforms were measured in the non-infarcted area of LV from rats with myocardial infarction (MI), without (H) or with (F) LV failure, and in control (C) rats. Basal, GTP- and forskolin-stimulated Mg2+- and Mn2+-dependent AC activities were also measured in F and C rats. Methods: Two- and six months after MI, steady-state AC mRNA concentrations were assessed by Northern blot analysis and RNase protection assay with isoform-specific cDNA and cRNA probes, respectively. AC activities were assessed on LV microsomal fractions using standard procedures. Results: Types V and VI, and types IV and VII were the major and minor AC mRNA isoforms in both the LVs of F and C rats. Two months after MI, no difference in LV type V or VI mRNA to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA ratios was observed in rats with H or F compared to C. Six months after MI, no difference in LV type V mRNA concentration was observed between the three rat groups, whether this level was normalized to GAPDH, poly-(A+) or 18S RNAs. In contrast, a 35% decrease in the type VI mRNA to poly-(A+) RNA ratio and a 29% decrease in the type VI mRNA to 18S RNA ratio was observed only in rats with F compared to C ( p

See article by Shinohara et al. [1] (pages 249–258) in this issue. Circulating antibodies, reacting with autoantigens in the vascular wall, are commonly harmless in healthy vessels but can turn pathogenic in the presence of vascular injury. The exact antigenic determinants and the role of such autoreactive antibodies remain unclear. In their present study appearing in this issue, Shinohara and coworkers [1] propose a xenogenic, vaccinal experimental approach in order to limit intimal proliferation in response to balloon-induced arterial wall injury in rabbits. Their observations suggest that the observed humoral response is responsible for the limitation of smooth muscle cell (SMC) migration and proliferation via an extracellular effect on the SMC response to growth factors and angiotensin II, as observed in vitro. While the involvement of autoreactive antibodies in human autoimmune diseases and their vascular targeting are beyond the scope of the present editorial, the observations of Shinohara and coworkers merit some comments. The authors [1] used a mixture of specific and non-specific cellular xenoantigens shared by cultured rat SMC rendered inert for immunization. They also used a homogenized rabbit SMC preparation, including membrane-exposed and intracellular antigens, for exploring the autoreactive determinants of the response. Therefore, a precise identification of the molecular targets of the humoral response is difficult to evaluate. The presence of auto-antibodies, within the intima in vivo, was … *Corresponding author. Email address: jbmichel{at}bichat.inserm.fr

Objective: The fibroblasts producing collagen are co-localized with inflammatory cells in myocardial fibrosis areas of spontaneously hypertensive rats, suggesting that collagen overproduction in this model may be modulated by inflammatory cells. The present study extends these observations to the Goldblatt model of hypertension in which the renin-angiotensin system is activated. Methods: Inflammatory cells were identified with monoclonal antibodies directed against macrophages (ED1+), T helper (CD4+) and cytotoxic lymphocytes (CD8+), and MHC class II-expressing cells (Ia+). The alkaline phosphatase-anti-alkaline phosphatase (APAAP) immunostaining technique was used. A new computer-assisted morphometric method was utilized to quantify the inflammatory infiltrate in each cardiac compartment with polarized-light microscopy. Cells responsible for the collagen synthesis were identified by in situ hybridization. The collagen content was estimated by morphometry on left ventricle sections stained with Sirius red, and by biochemical quantification of the hydroxyproline concentration. Results: Computer-assisted morphometry under polarized light was well suited to quantify inflammatory cells labeled by the APAAP technique. Inflammatory cells were co-localized with collagen-synthesizing fibroblasts. The main inflammatory cells were CD4+ lymphocytes>Ia+>ED1+>CD8+ cells. These cell densities were increased in hypertensive rats in all cardiac areas compared to control rats except for Ia+ cells which were concentrated in microscars. Macrophage density was correlated with plasma renin activity. The inflammatory cell density which best correlated with fibrosis was macrophage density, and which best correlated with systolic blood pressure was macrophage and T helper lymphocyte densities. Conclusions: One can speculate that the correlation between macrophage density and blood pressure as well as with plasma renin activity may indicate that angiotensins and/or elevation of blood pressure could participate in the initial signalling which may mobilize inflammatory cells. These inflammatory cells could promote fibrosis by releasing mediators such as growth factors or cytokines which act upon fibroblasts.

Objective: Inflammatory cells invade the fibrotic myocardium of spontaneously hypertensive rats at the same sites as where fibroblasts are produced. The role of these inflammatory cells in myocardial fibrogenesis was studied in the present work. Methods: The production and distribution of proteins that may be implicated in inflammation was examined by immunohistochemistry of sections of left ventricles from 1-month and 4-month renovascular hypertensive and age-matched control rats using antibodies against ICAM-1, LFA-1, TGFβ1, PDGF-A, T and H kininogens, IgG, IgM, C3, and C5b-9. Infiltrating inflammatory cells were phenotyped by immunohistochemistry. The TGFβ1 and PDGF-A mRNA levels were checked by RT-PCR. Results: Infiltrating cells were mainly T helper lymphocytes and macrophages, and there were more inflammatory cells in hypertensive rats than in control rats, localized especially around coronary arteries and in microscars. There were more ICAM-1 and LFA-1 in the ventricles of hypertensive than in control rats at 1 month, but the ICAM-1 expressions in hypertensive and control rats were similar at 4 months. TGFβ1 and PDGF-A mRNA steady states increased in 4-month hypertensive rats, but there was no labeling for TGFβ or PDGF by immunohistochemistry. There was only faint labeling for T and H kininogens, and it was not increased in hypertensive rats. There were deposits of IgM and C5b-9 only in hypertensive rats. Conclusion: Thus, inflammatory cells infiltrate the cardiac tissue of renovascular hypertensive rats as in the case of spontaneously hypertensive rats and these cells may use the ICAM-1/LFA-1 system to infiltrate, but neither TGFβ and PDGF-A, nor the kininogen system seem to be associated with cardiac fibrogenesis. Otherwise, the complement system could act as arteriosclerotic and/or leukocyte mobilizing factors.

Objectives: Angiotensin converting enzyme (ACE) activity in the plasma does not change significantly with hypertension in two-kidney, one-clip hypertensive (2K-1C) rats. However, heart ACE activity and mRNA increase with hypertension. We measured the ACE activity and mRNA in hypertrophied hearts at different times after clipping, and determined the cellular distribution of its increase in the left ventricle of 2K-1C hypertensive rats. Methods: Cardiac ACE activity was quantified in left and right ventricles using a radiolabeled synthetic ACE substrate, and ACE mRNA steady-state level was quantified by ribonuclease protection assay. Tissue localization of ACE in normal and hypertrophied hearts was determined by measuring ACE activity in isolated ventricular cells. In situ hybridization with a rat ACE cDNA and immunohistochemistry with a monoclonal anti-ACE antibody were used to identify tissue compartments producing ACE mRNA and protein. Results: The left ventricle was hypertrophied 2 weeks after clipping and remained hypertrophied at 12 weeks. Left ventricular ACE activity was significantly increased 2 and 4 weeks (3.2 ± 0.3 in 2K-1C vs. 1.7 ± 0.1 pmol/mg prot/min in sham-operated rat) after renal artery clipping, but not at 12 weeks. The right ventricle was slightly hypertrophied 4 weeks after clipping and remained hypertrophied at 12 weeks. Right ventricular ACE activity was significantly increased at 4 (6.7 ± 0.6 in 2K-1C vs. 3.1 ± 0.3 pmol/mg prot/min in sham-operated rat) and 12 weeks. ACE activity was not detectable in cardiomyocytes isolated by Percoll gradient. Neither was ACE mRNA detected in isolated cardiomyocytes, even after ACE mRNA amplification by RT-PCR. In contrast, ACE activity and mRNA were detected in pooled non-cardiomyocytic cells. Thus the increase in cardiac ACE activity associated with hypertension must be due to an increase in ACE expression by non-cardiomyocytic cells. In situ hybridization showed an autoradiographic signal for ACE mRNA over the endothelial cells of coronary arteries and over the interstitial spaces including pericoronary and fibrosis areas. Immunohistochemistry confirmed these data, showing ACE on endothelial cells and in pericoronary spaces with an increased signal in pericoronary and fibrosed areas in hypertensive hypertrophied left ventricle. Conclusion: Besides its usual endothelial expression, ACE is absent from cardiomyocytes and present in interstitial tissue, in the pericoronary spaces in normal tissue and more markedly in hypertrophied ventricles.

Objective: Low output heart failure induces abnormalities of endothelium dependent vasodilatation, but the mechanisms responsible for this remain unclear. As blood flow can alter endothelial cell function, in particular nitric oxide (NO) release, the activity of endothelium derived relaxing factor (EDRF) was investigated in a rat model of high output heart failure. Methods: The thoracic aorta upstream of an aorto-caval fistula in rats was submitted to hormonal changes (similar to those in heart failure) and to high blood flow (opposite to that found in low output heart failure). Functional and biochemical arterial properties were studied in aorto-caval fistula rats and in sham operated rats three months after operation. The vascular responses were studied by exposing aortic segments from fistula and sham operated rats to increasing concentrations of agonists. Aortic cyclic guanosine monophosphate (cGMP) concentration was assessed as an index of NO synthase activity. The effect of NO synthase blockade on functional and biochemical arterial properties was also studied. Results: Plasma atrial natriuretic factor (ANF) was increased in fistula rats compared to sham operated rats. The concentrations of acetylcholine or the calcium ionophore A23187 required to produce 10% and 50% maximum relaxation (EC, and EC50) were similar in the two groups. Relaxation in response to low concentrations of Sin-1 (an NO donor) was shifted rightwards in fistula rats and EC10 was greater than in the controls. The aortic cGMP concentration was higher in aorto-caval fistula rats than in sham operated rats (p=0.008). The differences between aorto-caval fistula rats and sham operated rats were probably the result of increased basal EDRF-NO release in the former, since NO synthase blockade abolished the differences in both aortic cGMP and the dose-response curve to Sin-1. Conclusions: The arterial wall upstream of a chronic aorto-caval fistula has increased cGMP content and hyposensitivity to Sin-1, which may be due to enhanced basal EDRF-NO release. These changes, strikingly different from those found in the low output heart failure, suggest that haemodynamic rather than neuroendocrine factors play a determinant role in the altered vasodilator response in heart failure. Cardiovascular Research 1993; 27 :1651-1656

Objectives: The state of the vasodilator systems in congestive heart failure is poorly defined. Plasma atrial natriuretic peptide is increased, whereas endothelium derived relaxing factor activity can be decreased. Atrial natriuretic peptide and endothelium derived relaxing factor both cause vascular relaxation by generating cyclic guanosine monophosphate (cGMP), by activating the particulate and the soluble guanylate cyclase, respectively. This study examines the biological effects of atrial natriuretic peptide and endothelium derived relaxing factor in experimental heart failure by assessing the plasma, urinary, and tissue concentrations of their common second messenger cGMP. Methods: Myocardial infarctions (n=31) were induced and sham operations (n=25) were performed on Wistar rats, and the rats were monitored for three months. Aortic and pulmonary cGMP contents were measured, as the aorta is mainly matrix and smooth muscle cells, and the lung is particularly rich in capillaries, hence in endothelial cells. The concentrations of the other second messenger cyclic adenosine monophosphate (cAMP) was also determined, as were those of cGMP dependent protein kinase in the arteries. Results: 17 of the 31 rats with myocardial infarction had oedema. The total heart weight to body weight ratio and the ratio of the myocardium haemodynamically upstream from the infarcted left ventricle to body weight were increased in proportion to the infarct size. Plasma atrial natriuretic peptide and plasma and urinary cGMP concentrations were increased in proportion to the degree of heart failure (p

Spontaneously hypertensive (SH) rats immunized with mouse renin produce anti-renin antibodies, responsible for down-modulation of blood pressure, associated with an infiltration of kidneys by mononuclear cells. In this work, anti-renin T cells from SH rats immunized with renin have been stimulated in vitro, and we have studied in vitro and in vivo their effect on anti-renin antibody production by normal syngeneic B cells. We show that, in vitro, renin-activated T cells induce a renin-specific antibody response without addition of exogenous renin. Anti-renin T cells injected into naive SH rats trigger normal B cells to secrete high amounts of monospecific anti-renin IgG antibodies as early as day 5. These antibodies interfere with the homeostasis of the renin-angiotensin system leading to the normalization of blood pressure without any nephritis. These results show that anti-renin B cells are either not tolerant per se or in a reversible state of anergy. Our results also suggest that anti-renin B cells constitutively express renin-derived peptides in such a way that they may be stimulated by activated anti-renin T cells; these cells express IL-4 mRNA indicating that IL-4 could play a role in the differentiation of B cells.

The beneficial effect of angiotensin-converting enzyme (ACE) inhibitors on myocardial mass and contractility in hypertension and, possibly, congestive heart failure (CHF) may be related to their ability to induce a decreased afterload. This has been assessed in four experimental models--renovascular hypertension, DOCA-salt hypertension, spontaneously hypertensive rats (SHR) and myocardial infarction (MI)--and in normotensive mature rats. In renovascular hypertension, ACE inhibitors normalized blood pressure as well as left ventricular hypertrophy and hypocontractility. In the DOCA-salt model, blockade of the renin-angiotensin system by ACE inhibitors did not decrease blood pressure and therefore had no effect on cardiac mass and contractility. In the SHR model, the arterial smooth muscle cell is functionally and structurally abnormal; as a result, cardiac overload led, over time, to a terminal, decompensated phase of CHF. ACE inhibitors, by decreasing blood pressure, reversed cardiac hypertrophy, hyperfibrosis and atrial natriuretic factor (ANF) oversecretion and prevented overload and time-induced CHF. In the MI model, ACE inhibitors decreased blood pressure and thereby decreased overload and reversed cardiac hypertrophy, hypocontractility, hyperfibrosis and ANF oversecretion. In normal ageing, heart function and structure are modified over time. ACE inhibitors, by blocking a 'normal' signal upstream, allowed a 'normal' effector system to decrease blood pressure and prevented the development of age-dependent cardiac hypertrophy.

Thoracic aortic aneurysm (TAA) refers to an abnormal progressive dilation of the aorta involving all 3 layers, histopathologically characterized by fibrillar extracellular matrix breakdown and the presence of large areas containing mucoid material. All this degradation is a result of the action of metalloproteinases activated via plasmin through its activators. Recently we showed: the action of the fibrinolytic system on the wall of the aorta with aneurysm, the presence of plasminogen/plasmin on the vascular smooth muscle cell (VSMC) surface and inside cytoplasmic vesicles, as well as the overexpression of protease nexin 1 (PN1). Increased expression of PN1 suggests a response of VSMCs to proteolysis by blocking the action of MMPs and thus preventing the advance of the disease. Our hypothesis is that PN-1 binds to plasmin and this complex is endocytosed via low-density lipoprotein receptor-related protein-1 (LRP-1), thus inactivating this serine-protease. This study aimed to verify the expression of LRP1 and to evaluate its possible interaction (co-location) with PN-1 and plasminogen/plasmin in the tunica media of normal and aneurysmal aortas. Sections of aortas from cases of TAA (n=4) and controls (n=4) were analyzed by immunoperoxidase and immunofluorescence to LRP-1, plasminogen/plasmin and PN-1. Sections of aortas with aneurysms showed high presence and co-localization between LRP1, plasminogen/plasmin and PN1. In contrast, normal aortas showed low presence of PN1, LRP1 and plasminogen/plasmin, it is not possible to observe a high co-location. These findings strengthen our hypothesis of endocytosis of PN1/plasmin complex via LRP1 and the modulating role of PN1 in thoracic aortic aneurysms.