Antioxidant/oxidant status and cardiac function in bradykinin B(1)- and B(2)-receptor null mice.

Kinin-vasoactive peptides activate two G-protein-coupled receptors (R), B(1)R (inducible) and B(2)R (constitutive). Their complex role in cardiovascular diseases could be related to differential actions on oxidative stress. This study investigated impacts of B(1)R or B(2)R gene deletion in mice on the cardiac function and plasma antioxidant and oxidant status. Echocardiography-Doppler was performed in B(1)R (B(1)R(-/-)) and B(2)R (B(2)R(-/-)) deficient and wild type (WT) adult male mice. No functional alteration was observed in B(2)R(-/-) hearts. B(1)R(-/-) mice had significantly lowered fractional shortening and increased isovolumetric contraction time. The diastolic E and A waves velocity ratio was similar in all mice groups. Thus B(1)R(-/-) mice provide a model of moderate systolic dysfunction, whereas B(2)R(-/-) mice displayed a normal cardiac phenotype. Plasma antioxidant capacity (ORAC) was significantly decreased in both B(1)R(-/-) and B(2)R(-/-) mice whereas the vitamin C levels were decreased in B(2)R(-/-) mice only. Plasma ascorbyl free radical was significantly higher in B(1)R(-/-) compared to WT and B(2)R(-/-) mice. Therefore, the oxidative stress index, ascorbyl free radical to vitamin C ratio, was increased in both B(1)R(-/-) and B(2)R(-/-) mice. Hence, B(1)R and B(2)R deficiency are associated with increased oxidative stress, but there is a differential imbalance between free radical production and antioxidant defense. The interrelationship between the differential B(1)R and B(2)R roles in oxidative stress and cardiovascular diseases remain to be investigated.

[Inflammasome and cardiovascular diseases]. / Rôle de l'inflammasome dans les pathologies cardiovasculaires.

NOD-like receptors (NLRs) constitute a recently identified family of intracellular pattern recognition receptors which contains more than 20 members in mammals. Some of the NLRs, the NALP subfamily, constituted from 14 members, many of them without actual identified role, form multiproteic complex known as inflammasome, that initiate inflammation by processing inactive pro-caspase-1 to its active form, allowing the cleavage and subsequent activation of pro-IL-1ß and pro-IL-18. We review the identified roles of NLRs in pathologies and argue for the role of inflammasome in the development of cardiovascular diseases. The atherogenic cytokines IL-1ß and IL-18 are matured in NLRPs inflammasomes. Immunocytochemistry shows that Nlrp3 inflammasome is expressed in plaques, upregulated and activated in the CD11b(+)Gr1(high) atherosclerosis-prone monocyte subset and modulated by oxLDL in murine macrophages. These results provide an unexpected role for Nlrp3 inflammasome in atherosclerosis.

Potential role of the neuropeptide CGRP in the induction of differentiation of rat hepatic portal vein wall.

The media of the rat hepatic portal vein is composed of an internal circular muscular layer (CL) and an external longitudinal muscular layer (LL). These two perpendicular layers differentiate progressively from mesenchymal cells within the first month after birth. In this paper, we studied the development of calcitonin gene-related peptide (CGRP) innervation during post-natal differentiation of the vessel. We show that CGRP innervation is already present around the vessel at birth in the future adventitia but far from the lumen of the vessel. Progressively, CGRP immunoreactive fibers reached first LL then CL. CL by itself become only innervated at day 14 after birth. This corresponds to the time at which thick filaments (myosin) are visible in electron microscopy and desmin visualisable by immunocytochemistry. Furthermore, we provide evidence by autoradiography, that binding sites for CGRP are transiently expressed on the portal vein media at day 1 and 14 after birth. Vascular smooth muscle cells were transfected with constructs containing promoters for desmin or smooth muscle myosin heavy chain (smMHC). CGRP treatment of the cells significantly increased the expression of smMHC. Overall these results suggest that CGRP can potentially influence the differentiation of smooth muscle cells from the vessel wall.

Calcitonin gene-related peptide partly protects cultured smooth muscle cells from apoptosis induced by an oxidative stress via activation of ERK1/2 MAPK.

Oxidative stress induced by a glucose/glucose oxidase (G/GO) generator system dose-dependently decreased the viability of cultured vascular smooth muscle cells (VSMC) as estimated by MTT assay. Cell death was induced in 40% of cells exposed to 0.2 IU/ml of the free radical generating mixture. Annexin-V labeling, Hoechst staining together with DNA laddering demonstrated that apoptosis was responsible for this cell loss. Pretreatment of the cells with 10(-8) M calcitonin gene-related peptide (CGRP) significantly attenuated the damaging effect of the oxidative stress. Indeed, cell viability was estimated to be 80% in CGRP-treated group, instead of 60% in absence of CGRP treatment. This protective effect of CGRP was antagonized by 8-37 CGRP, an antagonist of CGRP-1 receptors, whereas it was not reproduced by amylin, a CGRP analogue. As indicated by the reduction in Hoechst staining and in DNA laddering, CGRP prevented the onset of apoptosis. We also demonstrated that the peptide significantly up-regulated the activation of ERK1/2 and P38 kinases. Inhibitors of the kinases prevented the protective effect of CGRP. We conclude that CGRP antagonizes oxidative stress-induced apoptosis by up-regulating MAP kinase activation and that activation of these kinases was necessary to protection.

Effect of in vivo heart irradiation on coronary reactivity in the rat.

The frequent exposure of the heart to radiation during thoracic tumor radiotherapy often results in chronic impairment of myocardial function. The aim of the present investigation was to evaluate the effect of irradiation on coronary vascular tone in rat hearts exposed in vivo to a single dose of 20 Gy gamma rays. The ability of rat hearts to respond to changes in coronary reactivity was analyzed 1, 15, 30 and 60 days following cardiac irradiation, using the Langendorff model, after perfusion of either L-nitro-arginine (LNA), an inhibitor of nitric oxide synthetase or SIN 1, a nitric oxide donor drug. LNA-induced vasoconstriction and SIN 1-induced vasodilation were lost respectively 15 days and 30 days after irradiation, and associated with smooth muscle cell alterations observed in microscopy, but without any changes in myocardial MDA levels. Thus, our results suggest that 1) endothelium may represent an early and specific radiation target, characterized by radiation-induced vascular tone dysfunctions, with no detectable microscopical changes; 2) alterations are progressive, resulting first from endothelial damage, followed by smooth muscle cell injuries. In conclusion, a local cardiac irradiation induced cellular dysfunction, characterized by a loss of coronary reactivity without changes of the lipid peroxidation index in the hearts.

The neuropeptide calcitonin gene-related peptide differently modulates proliferation and differentiation of smooth muscle cells in culture depending on the cell type.

Calcitonin gene-related peptide (CGRP) is a neuropeptide present around vasculature very early during development, when smooth muscle cells (SMC) are still proliferating and not yet totally differentiated. We investigated the effects of CGRP on proliferation and differentiation of SMC in culture; 10(-7) M CGRP added in the medium of cultured smooth muscle cells every 2 days did not significantly changed cells growth rate in 1% FCS. At the opposite, this treatment modulated proliferation of cells grown in 10% FCS medium. Two distinct populations of SMC with different growth rates were obtained from our primary cultures. SMC which proliferated slowly in the presence of 10% fetal calf serum (FCS) had growth rates positively influenced by CGRP. The quantity of alpha-smooth actin expressed by these cells was not influenced by the peptide. On the contrary, SMC which proliferated more rapidly in 10% FCS medium had growth rate inhibited by CGRP. In these cells, CGRP significantly reduced the amount of expressed alpha-smooth actin, an index of SMC differentiation. In both cases, the peptide significantly increased the level of mRNA for all the actin genes. In the light of this dual role of CGRP, it can be presumed that this peptide controls smooth muscle cells proliferation and differentiation in vivo and could thus regulate the homeostasis of the vessel wall.

Modification of the rat aortic wall during ageing; possible relation with decrease of peptidergic innervation.

Structural changes of the male rat aorta were followed from birth to old age in male and female rats. In males, the vessel media width and area progressively increase concomitantly with a decrease of nuclei density during ageing, suggesting an hypertrophy of the smooth muscle cells. These correlations were however not evidenced in females. TUNEL-positive cells were found in media of 4 and 6 months in both sexes, mainly on the luminal side and in the adventitia. When biochemical markers were investigated with immunohistochemistry, media was uniformly stained by the anti-vimentin and anti-alpha-smooth actin at all stages investigated. On the contrary, the surface of media stained with anti-desmin decreased during ageing, especially on the luminal side. As observed with electron microscopy, with ageing the endothelium is replaced by small cells with pseudopodia adhering to the vestigial elastic lamina and infiltrating into the extracellular matrix left after the disappearance of smooth muscle cells. In addition, in the older rats (25-29 months) the elastic laminae are completely disorganised. Hypertrophy of the smooth muscle cells was confirmed by this approach. In parallel to this study, perivascular peptidergic innervation was stained with antibodies against calcitonin gene-related peptide (CGRP), substance P (SP), neuropeptide Y (NPY), and vasoactive intestinal polypeptide (VIP) at different ages during the whole life of rats. These peptides are present in stages younger than 6 months, then gradually disappear. In one year animals and older, the peptidergic innervation has totally disappeared. We discuss the possible role of peptidergic innervation in the control of the vessel wall cellular stability during ageing.

Connexin43 gap junction levels during development of the thoracic aorta are temporally correlated with elastic laminae deposition and increased blood pressure.

A characteristic property of the vascular smooth muscle cell is its ability to modulate between a contractile phenotype, responsible for control of vascular tone, through to a synthetic phenotype, capable of migration and synthesis of extracellular matrix molecules. Smooth muscle cells are coupled by gap junctions, the membrane structures which permit direct intercelluar passage of ions and small molecules, and which play a role both in electrical coupling and intercellular communication during patterning and development. We have previously found that connexin43 type gap junction expression is upregulated in the synthetic phenotype smooth muscle cell in vitro and during atherosclerotic plaque formation in human coronary arteries. On the basis of immunohistochemical labelling, confocal laser scanning microscopy and digital image analysis, we now report that relatively high levels of connexin43 are expressed during development of the rat thoracic aorta, temporally correlating with reported periods of smooth muscle cell proliferation and secretion of elastic laminae. A major peak in expression occurs at seven days post-natal, with a second less pronounced peak at 72 days post-natal. The principal peak in gap junction levels appears to coincide with increased post-natal blood pressure and aorta media thickening. The amount of gap junction labelling falls off to normal adult levels as the smooth muscle cells modulate towards the contractile phenotype and growth is completed. The results indicate an association between direct cell-to-cell communication and synthetic phenotype smooth muscle cell activity during aortic growth and patterning.

CGRP innervation and receptors during aging of male and female hepatic rat portal veins.

Calcitonin gene-related peptide (CGRP) innervation and binding sites were studied on hepatic portal veins of male and female rats from 19 days to 22 months of age. CGRP containing nerve fibers were present both in adventitial and medial nerve plexuses, closely apposing to or penetrating into the muscular layers. The density of CGRP innervation was estimated on whole mount preparations and compared during aging. In females, aging did not affect the number of fibers per unit length, although the vessel circumference decreased after 6 months of age. In males, the vessel circumference remained constant during aging, while the density of innervation significantly decreased. Whatever the age or sex of the animals was, no CGRP binding sites were found on portal veins sections by autoradiography. CGRP had no effect on spontaneous contractions of perfused portal veins. The difference observed in the evolution of CGRP innervation density between males and females suggests that CGRP innervation in the rat portal vein may be influenced by gonadal steroids during aging.

Rat common bile duct: structure, pharmacological responsiveness, CGRP innervation, and binding sites.

The wall of the rat common bile duct (CBD) consists of several epithelial ducts embedded in connective tissue which contains some regions with cells weakly stained by an antibody against alpha smooth muscle actin. The hepatic side (HS) is more vascularized than the duodenal side (DS). Calcitonin gene-related peptide (CGRP)-like immunoreactivity is present in nerve fibres penetrating deeply into the CBD wall. On whole-mount preparations, CGRP innervation is mainly associated with blood vessels in the HS, whereas it forms a wide meshed network independent of vasculature in the DS. Abundance of CGRP innervation was compared between both sexes and at different ages. No differences were found in the total number of fibres between males and females except at 4 months of age, when males had statistically more abundant innervation than females. However, during aging, while the abundance of innervation (fibers/mm) remained stable in both HS and DS in females, it significantly decreased in males. Autoradiography demonstrated the presence of 125I-CGRP binding sites in the rat CBD. In vitro, 30% of HS strips showed spontaneous rhythmic contractions but all the strips (autocontractile or not) contracted dose dependently in response to acetylcholine (Ach) or substance P (SP). However, DS strips were neither autocontractile nor responsive to Ach or SP. Perfusion of all strips with 10(-7) M CGRP produced no effects nor influenced Ach- or SP-induced contractions.

Effects of ingested phytoecdysteroids in the female soft tick Ornithodoros moubata.

The effects of the ingestion of some phytoecdysteroids were studied in the soft tick Ornithodoros moubata. Supernumerary moulting and malformations of first leg pairs were obtained with 22-oxo-20-hydroxy-ecdysone, 20-hydroxyecdysone-22-acetate, and 20-hydroxyecdysone-22-benzoate. Egg-yield was reduced with 20-hydroxyecdysone-22-acetate and carthamosterone. Finally, drying-out of eggs was observed with carthamosterone and 22-deoxy-20,26-dihydroxyecdysone. In addition, we demonstrated that there is a correlation between the number of completed gonotrophic cycles and the impossibility of inducing supernumerary moulting.

Ecdysteroid titre and metabolism and cuticle deposition during embryogenesis of the ixodid tick Amblyomma hebraeum (Koch).

Three embryonic cuticles are formed before larval cuticle deposition during embryonic development of Amblyomma hebraeum. The quantity of radioimmunoassay-positive material varied between 50 and 200 pg ecdysone equivalents per mg, but no significant peaks were detected. Maternally incorporated [3H]-20-hydroxyecdysone and [3H]-ecdysone contained in freshly laid eggs appear to be conjugated to C-22 fatty acid esters and 3 alpha epimers of those esters, and, thus, appear doubly inactivated. In addition, ecdysone is converted to an unknown product called 2'. The role of these maternally derived ecdysteroids is unknown.

Cytoskeletal features in longitudinal and circular smooth muscles during development of the rat portal vein.

Immunohistochemistry of alpha-smooth muscle actin and desmin, two markers of smooth muscle cell differentiation, and electron-microscopic observation of thick filaments of myosin were performed on the media of the developing rat hepatic portal vein to gain insights into the chronology of differentiation of its longitudinal and circular smooth muscles. In accordance with the ultrastructural distribution of thin filaments, staining of alpha-smooth muscle actin is lightly positive in the myoblasts at postnatal day 1 and then extends in probably all muscle cells of the developing vessel. Desmin, which appears later than alpha-smooth muscle actin in the two muscles, is distributed throughout the longitudinal layer at day 8, whereas the first arrangements of thick filaments are detectable in most longitudinal muscle cells; at this stage, desmin and thick filaments are absent from the poorly differentiated circular muscle cells. The longitudinal muscle cells differentiate in a strikingly synchronized way from day 8 onwards, conferring a homogeneous structure to the developing and mature longitudinal layer. Several desmin-positive cells and a heterogeneous distribution of thick filaments occur in the circular muscle at day 14; the subsequent extension of these filaments in this layer results in a persisting heterogeneous distribution in the young 7-week-old adult. Many features of the mature smooth muscle cells are established within the third week in the longitudinal muscle, approximately one week before those of the circular layer. These results are consistent with the function of the longitudinal muscle as a spontaneously contractile smooth muscle unit, and emphasize the need for its fast maturation to fulfil its major role in the control of portal blood flow.

Calcitonin gene-related peptide innervation and binding sites in rat aorta during development.

Indirect immunohistochemistry performed on whole mounts of arch and thoracic part of the rat aorta at six developmental stages (from embryonic day 17 to 6 months, in males and females) revealed that calcitonin gene-related peptide (CGRP) innervation is highest in the arch. The highest density of innervation is found at the three first postnatal ages investigated (day 1, day 3 and 5 weeks; 2.6 +/- 0.6 intercepts/mm in the arch at 1 day); however, all values are low compared to other arteries. The innervation grows from a few short isolated fibres in the embryo to a more complex meshwork in older animals. No striking differences were noticed between males and females. Autoradiographic studies were performed on serial sections at several levels of the aorta but did not reveal binding sites for CGRP in the vascular wall. This might be due to the technique which does not allow visualization of low density of binding sites, or to binding sites of weak affinity. We discuss the possible importance of CGRP in rat aortic smooth muscle development.

Postnatal development of the rat portal vein: correlation with occurrence of peptidergic innervation.

The portal vein of the rat is immature at birth, and is composed of an endothelium surrounded by undifferentiated cells of mesenchymal origin. Three days after birth, these cells have begun to differentiate and aggregate around the lumen to form two separate layers of perpendicularly oriented myoblasts, while a rich calcitonin gene-related peptide (CGRP) innervation is present around the vessel. In the internal circular muscle layer of the media myofibrils first develop on the endothelial side of the myoblasts, and then progressively reach the other side. In the longitudinal muscular layer of the media, which is separated from the circular layer by a connective lamina as early as 3 days after birth, myofibrils develop randomly in the cells. At the time of the enlargement of the longitudinal layer, long close contacts and intermediate junctions between external myoblasts and adventitial fibroblast-like cells were noted, suggesting that recruitment of this cell type is necessary for the maturation of the vessel wall. At about 28 days, the vein has reached its final structure and the smooth muscle cells are fully differentiated. The dense CGRP perivascular innervation already present at birth persists for the first 14 days of postnatal life when most of the cells have not yet acquired their complete contractile differentiation and are still capable of division. This innervation decreases transiently between 15-17 days, when the vessel acquires its spontaneous contractile activity, then rises to a peak between 20 and 25 days, and falls again. CGRP innervation, which is very scarce at 28 days, slowly increases during the peripubescent stage, by which time the adult structure of the vessel is established. Similar fluctuations in the density of peptidergic innervation were observed for substance P and neuropeptide Y, although these peptides were not yet present at birth and occurred only after 5 days. Vasoactive intestinal polypeptide- and bombesin-immunoreactive fibres were not found at any stage investigated. In addition to a description of the different cell-to-cell contacts which could play a role in the maturation of the vessel wall, we discuss the possible implication of the different peptides in the differentiation, maturation or maintenance of the vessel wall.

Expression of connexin43 gap junctions between cultured vascular smooth muscle cells is dependent upon phenotype.

The smooth muscle cell is the predominant cell type of the arterial media. In the adult vascular system, smooth muscle cells are found primarily in the contractile phenotype, but following injury or during atherosclerotic plaque formation the secretory synthetic phenotype is expressed. Recently it has been shown that gap junction connexin43 messenger RNA levels are six times higher in cultured smooth muscle cells in the synthetic phenotype than in intact aorta. We have modulated rabbit aortic smooth muscle cells in culture between the synthetic phenotype and one resembling the contractile phenotype, and correlated gap junction expression with phenotype. A dual labelling technique with antibodies against smooth muscle myosin and a synthetic peptide constructed to match a portion of the connexin43 gap junction protein was used for these experiments. Gap junctions are numerous between synthetic phenotype cells but few are observed between contractile cells. Rat aortic smooth muscle cells were also cultured and the growth and structure of gap junctions followed in the synthetic phenotype by use of freeze-fracture electron microscopy and immunohistochemical techniques. Junctional plaques are similar in structure to those observed in cardiac muscle, their size and number increasing with time in culture. The increased numbers of gap junctions between synthetic phenotype smooth muscle cells may be important during vessel development, following injury, or in atherosclerotic plaque formation.

Metabolism of [3H]-ecdysone in embryos and larvae of the tick Ornithodoros moubata.

The fate of [3H]-ecdysone ([3H]-E) was investigated in hanging drop cultures of embryos and larvae of the tick Ornithodoros moubata using HPLC. The hormone was metabolized more slowly during described periods of increasing endogenous ecdysteroid (ES) titers than during periods of low titers except for young embryos. Three different classes of metabolites were produced: 1) apolar products (AP) corresponding to C-22 fatty acid ester conjugates of E and, in some cases, of 20-hydroxyecdysone (20E), 2) unidentified polar products (PP) more polar than E, one peak of which had the same retention times as 20,26-dihydroxyecdysone, and finally, 3) 20E verified by comigration of cold standards on RP-18 and silica columns. Hydroxylation of E to 20E first became evident in cultures of 2 day old embryos and was present in all cultures of older animals. Highest production of free 20E occurred during increasing endogenous ES titers in embryos and during the ES peak in larvae. Conjugation of E to AP occurred in all stages investigated, but was more pronounced during periods of low endogenous ES titers, and may correspond to a detoxification mechanism. In contrast, PP were produced during high 20E production in embryos and during periods of high and decreasing endogenous titers in larvae.

Occurrence of CGRP receptors on dedifferentiated smooth muscle cells from media of rat thoracic aorta.

Autoradiographic studies with [125I]-CGRP did not demonstrate receptors on sections from two different regions of 5-week-old rat aorta (of both sexes). By contrast, cultured smooth muscle cells isolated from similar aortic media and passaged 5 to 7 times exhibited specific binding.

An electron-microscopic study of smooth muscle cell dye coupling in the pig coronary arteries. Role of gap junctions.

Arterial smooth muscles behave like a syncytium, since they are electrically coupled. It is generally assumed that electrical coupling and dye coupling are mediated by gap junctions. No gap junctions could be detected by transmission electron microscopy in media of coronary arteries. We looked for the presence of gap junction protein in vascular smooth muscle by immunohistochemistry with light microscopy. Immunohistologically detectable connexin is expressed by smooth muscle cells of the media of pig coronary arteries, where staining occurs as a discrete punctation. We investigated the dye coupling in strips of pig coronary artery. The fluorescent dye lucifer yellow was microiontophoretically injected into a smooth muscle cell through an intracellular microelectrode. The dye was visualized on the entire strip, then on semithin sections with a fluorescence microscope, and at the ultrastructural level by using an anti-lucifer yellow antibody revealed by the protein A-gold technique. In all the tissues examined, the cells were dye-coupled. We conclude that in arterial media the smooth muscle cells are dye-coupled, despite the absence of detectable gap junctions by transmission electron microscopy, and suggest that dye coupling could occur via isolated gap junction channels.