Increased vascular and uteroplacental matrix metalloproteinase-1 and -7 levels and collagen type I deposition in hypertension in pregnancy: role of TNF-α.

Preeclampsia is a pregnancy-related disorder manifested as maternal hypertension in pregnancy (HTN-Preg) and fetal growth restriction. Placental ischemia could be an initiating event that leads to abnormal vascular and uteroplacental remodeling in HTN-Preg; however, the molecular targets and intermediary mechanisms involved are unclear. We tested the hypothesis that placental ischemia could target vascular and uteroplacental matrix metalloproteinases (MMPs) through an inflammatory cytokine-mediated mechanism. MMP levels and distribution were measured in the aorta, uterus, and placenta of normal pregnant (Preg) rats and pregnant rats with reduced uterine perfusion pressure (RUPP). Maternal blood pressure was higher and the litter size and pup weight were lower in RUPP compared with Preg rats. Gelatin zymography showed prominent uterine MMP-2 and MMP-9 activity that was dependent on the amount of loaded protein. At saturating protein loading, both gelatin and casein zymography revealed two additional bands corresponding to MMP-1 and MMP-7 that were greater in the aorta, uterus, and placenta of RUPP compared with Preg rats. Western blots and immunohistochemistry confirmed increased MMP-1 and MMP-7 in the aorta, uterus, and placenta of RUPP versus Preg rats. The levels of MMP-1 and MMP-7 substrate collagen type I were greater in tissues of RUPP compared with Preg rats. In organ culture, TNF-α increased MMP-1 and MMP-7 in the aorta, uterus, and placenta of Preg rats, and a TNF-α antagonist prevented the increases in MMPs in tissues of RUPP rats. Thus, placental ischemia, possibly through TNF-α, increases vascular and uteroplacental MMP-1 and MMP-7, which, in turn, alter collagen deposition and cause inadequate tissue remodeling in HTN-Preg. Cytokine antagonists may reverse the increase in MMP-1 and MMP-7 expression/activity and, in turn, restore proper vascular and uteroplacental remodeling in HTN-Preg and preeclampsia.NEW & NOTEWORTHY The molecular mechanisms of preeclampsia are unclear, making it difficult to predict, prevent, or manage the pregnancy-associated disorder. This study showed that placental ischemia, possibly through the release of TNF-α, causes increases in the levels of matrix metalloproteinase (MMP)-1 and MMP-7, which could alter collagen deposition and cause inadequate uteroplacental and vascular remodeling in hypertension in pregnancy. The data suggest that targeting MMP-1 and MMP-7 and their upstream modulators, such as TNF-α, could provide a new approach in the management of hypertension in pregnancy and preeclampsia.

Increased Atrial ß-Adrenergic Receptors and GRK-2 Gene Expression Can Play a Fundamental Role in Heart Failure After Repair of Congenital Heart Disease with Cardiopulmonary Bypass.

Surgeries to correct congenital heart diseases are increasing in Brazil and worldwide. However, even with the advances in surgical techniques and perfusion, some cases, especially the more complex ones, can develop heart failure and death. A retrospective study of patients who underwent surgery for correction of congenital heart diseases with cardiopulmonary bypass (CPB) in a university tertiary-care hospital that died, showed infarction in different stages of evolution and scattered microcalcifications in the myocardium, even without coronary obstruction. CPB is a process routinely used during cardiac surgery for congenital heart disease. However, CPB has been related to increased endogenous catecholamines that can lead to major injuries in cardiomyocytes. The mechanisms involved are not completely understood. The aim of this study was to evaluate the alterations induced in the ß-adrenergic receptors and GRK-2 present in atrial cardiomyocytes of infants with congenital heart disease undergoing surgical repair with CPB and correlate the alterations with functional and biochemical markers of ischemia/myocardial injury. The study consisted of right atrial biopsies of infants undergoing surgical correction in HC-FMRPUSP. Thirty-three cases were selected. Atrial biopsies were obtained at the beginning of CPB (group G1) and at the end of CPB (group G2). Real-time PCR, Western blotting, and immunofluorescence analysis were conducted to evaluate the expression of ß1, ß2-adrenergic receptors, and GRK-2 in atrial myocardium. Cardiac function was evaluated by echocardiography and biochemical analysis (N-terminal pro-brain natriuretic peptide (NT-ProBNP), lactate, and cardiac troponin I). We observed an increase in serum lactate, NT-proBNP, and troponin I at the end of CPB indicating tissue hypoxia/ischemia. Even without major clinical consequences in cardiac function, these alterations were followed by a significant increase in gene expression of ß1 and ß2 receptors and GRK-2, suggesting that this is one of the mechanisms responsible for the exacerbated response of cardiomyocytes to circulating catecholamines. These alterations could explain the irreversible myocardial damage and lipid peroxidation of membranes classically attributed to catecholamine excess, observed in some infants who develop heart failure and postoperative death. Although other factors may be involved, this study confirms that CPB acts as a potent inducer of increased gene expression of ß- adrenergic receptors and GRK-2, making the myocardium of these infants more susceptible to the effects of circulating endogenous catecholamines, which may contribute to the development of irreversible myocardial damage and death.

Adaptive increases in expression and vasodilator activity of estrogen receptor subtypes in a blood vessel-specific pattern during pregnancy.

Normal pregnancy is associated with adaptive hemodynamic, hormonal, and vascular changes, and estrogen (E2) may promote vasodilation during pregnancy; however, the specific E2 receptor (ER) subtype, post-ER signaling mechanism, and vascular bed involved are unclear. We tested whether pregnancy-associated vascular adaptations involve changes in the expression/distribution/activity of distinct ER subtypes in a blood vessel-specific manner. Blood pressure (BP) and plasma E2 were measured in virgin and pregnant (day 19) rats, and the thoracic aorta, carotid artery, mesenteric artery, and renal artery were isolated for measurements of ERα, ERß, and G protein-coupled receptor 30 [G protein-coupled ER (GPER)] expression and tissue distribution in parallel with relaxation responses to E2 (all ERs) and the specific ER agonist 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)-tris-phenol (PPT; ERα), diarylpropionitrile (DPN; ERß), and G1 (GPER). BP was slightly lower and plasma E2 was higher in pregnant versus virgin rats. Western blots revealed increased ERα and ERß in the aorta and mesenteric artery and GPER in the aorta of pregnant versus virgin rats. Immunohistochemistry revealed that the increases in ERs were mainly in the intima and media. In phenylephrine-precontracted vessels, E2 and PPT caused relaxation that was greater in the aorta and mesenteric artery but similar in the carotid and renal artery of pregnant versus virgin rats. DPN- and G1-induced relaxation was greater in the mesenteric and renal artery than in the aorta and carotid artery, and aortic relaxation to G1 was greater in pregnant versus virgin rats. The nitric oxide synthase inhibitor N(ω)-nitro-l-arginine methyl ester with or without the cyclooxygenase inhibitor indomethacin with or without the EDHF blocker tetraethylammonium or endothelium removal reduced E2, PPT, and G1-induced relaxation in the aorta of pregnant rats, suggesting an endothelium-dependent mechanism, but did not affect E2-, PPT-, DPN-, or G1-induced relaxation in other vessels, suggesting endothelium-independent mechanisms. E2, PPT, DPN, and G1 caused relaxation of Ca(2+) entry-dependent KCl contraction, and the effect of PPT was greater in the mesenteric artery of pregnant versus virgin rats. Thus, during pregnancy, an increase in ERα expression in endothelial and vascular smooth muscle layers of the aorta and mesenteric artery is associated with increased ERα-mediated relaxation via endothelium-derived vasodilators and inhibition of Ca(2+) entry into vascular smooth muscle, supporting a role of aortic and mesenteric arterial ERα in pregnancy-associated vasodilation. GPER may contribute to aortic relaxation while enhanced ERß expression could mediate other genomic vascular effects during pregnancy.

Estrogen receptor subtypes mediate distinct microvascular dilation and reduction in [Ca2+]I in mesenteric microvessels of female rat.

Estrogen interacts with estrogen receptors (ERs) to induce vasodilation, but the ER subtype and post-ER relaxation pathways are unclear. We tested if ER subtypes mediate distinct vasodilator and intracellular free Ca(2+) concentration ([Ca(2+)]i) responses via specific relaxation pathways in the endothelium and vascular smooth muscle (VSM). Pressurized mesenteric microvessels from female Sprague-Dawley rats were loaded with fura-2, and the changes in diameter and [Ca(2+)]i in response to 17ß-estradiol (E2) (all ERs), PPT (4,4',4''-[4-propyl-(1H)-pyrazole-1,3,5-triyl]-tris-phenol) (ERα), diarylpropionitrile (DPN) (ERß), and G1 [(±)-1-[(3aR*,4S*,9bS*)-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro:3H-cyclopenta(c)quinolin-8-yl]-ethanon] (GPR30) were measured. In microvessels preconstricted with phenylephrine, ER agonists caused relaxation and decrease in [Ca(2+)]i that were with E2 = PPT > DPN > G1, suggesting that E2-induced vasodilation involves ERα > ERß > GPR30. Acetylcholine caused vasodilation and decreased [Ca(2+)]i, which were abolished by endothelium removal or treatment with the nitric oxide synthase blocker Nω-nitro-l-arginine methyl ester (L-NAME) and the K(+) channel blockers tetraethylammonium (nonspecific) or apamin (small conductance Ca(2+)-activated K(+) channel) plus TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole) (intermediate conductance Ca(2+)-activated K(+) channel), suggesting endothelium-derived hyperpolarizing factor-dependent activation of KCa channels. E2-, PPT-, DPN-, and G1-induced vasodilation and decreased [Ca(2+)]i were not blocked by L-NAME, TEA, apamin plus TRAM-34, iberiotoxin (large conductance Ca(2+)- and voltage-activated K(+) channel), 4-aminopyridine (voltage-dependent K(+) channel), glibenclamide (ATP-sensitive K(+) channel), or endothelium removal, suggesting an endothelium- and K(+) channel-independent mechanism. In endothelium-denuded vessels preconstricted with phenylephrine, high KCl, or the Ca(2+) channel activator Bay K 8644 (1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridinecarboxylic acid methyl ester), ER agonist-induced relaxation and decreased [Ca(2+)]i were with E2 = PPT > DPN > G1 and not inhibited by the guanylate cyclase inhibitor ODQ [1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one], and showed a similar relationship between decreased [Ca(2+)]i and vasorelaxation, supporting direct effects on Ca(2+) entry in VSM. Immunohistochemistry revealed ERα, ERß, and GPR30 mainly in the vessel media and VSM. Thus, in mesenteric microvessels, ER subtypes mediate distinct vasodilation and decreased [Ca(2+)]i (ERα > ERß > GPR30) through endothelium- and K(+) channel-independent inhibition of Ca(2+) entry mechanisms of VSM contraction.

Interference of doxycycline pretreatment in a model of abdominal aortic aneurysms.

BACKGROUND: Abdominal aortic aneurysm (AAA) is characterized by chronic inflammation and degradation of the extracellular matrix, mediated by matrix metalloproteinases (MMPs). Doxycycline has been reported to control the progression of AAA by regulation of MMP. We hypothesized that doxycycline pretreatment in a rat model of AAA would cause reduction in gelatinolytic activity of MMP-2 and -9 and the inflammatory response in the wall of an aneurysm, consequently decreasing the formation and development of AAAs. METHODS: Male Wistar rats were divided into the following four groups: aneurysm (A); control (C); aneurysm+doxycycline (A+D) and control+doxycycline (C+D), with 24 animals per group subdivided into n=6 animals at different time points [1, 3, 7, and 15 days postsurgery (dps)]. The (A) and (A+D) groups simultaneously received the injury and extrinsic stenosis of the aortic wall. The (C) and (C+D) groups received sham operation. The treated animals received doxycycline via gavage (30 mg/kg/day) from 48 h before surgery until the end of experiment. At 1, 3, 7, and 15 dps, the animals were euthanized, and the aortas were collected for morphological analyses, immunohistochemistry, and zymography. RESULTS: The animals from the (A) group developed AAAs. However, the animals treated with doxycycline showed a 85% decrease in AAA development, which was associated with a large reduction in gelatinolytic activity of MMP-2 and -9, and decreased inflammatory response (P<.05). CONCLUSIONS: These results suggest that pretreatment with doxycycline before surgery inhibited the activity of MMP-2 and -9, as well as the inflammatory response, and may play an important role in the prevention of the development of AAAs.

Downregulation of microvascular endothelial type B endothelin receptor is a central vascular mechanism in hypertensive pregnancy.

Preeclampsia is a pregnancy-related disorder characterized by hypertension with an unclear mechanism. Studies have shown endothelial dysfunction and increased endothelin-1 (ET-1) levels in hypertensive pregnancy (HTN-Preg). ET-1 activates endothelin receptor type-A in vascular smooth muscle to induce vasoconstriction, but the role of vasodilator endothelial endothelin receptor type-B (ETBR) in the changes in blood pressure (BP) and vascular function in HTN-Preg is unclear. To test whether downregulation of endothelial ETBR expression/activity plays a role in HTN-Preg, BP was measured in normal pregnancy (Norm-Preg) rats and rat model of HTN-Preg produced by reduction of uteroplacental perfusion pressure (RUPP), and mesenteric microvessels were isolated for measuring diameter, [Ca(2+)]i, and endothelin receptor type-A and ETBR levels. BP, ET-1- and potassium chloride-induced vasoconstriction, and [Ca(2+)]i were greater in RUPP than in Norm-Preg rats. Endothelium removal or microvessel treatment with ETBR antagonist BQ-788 enhanced ET-1 vasoconstriction and [Ca(2+)]i in Norm-Preg, but not RUPP, suggesting reduced vasodilator ETBR in HTN-Preg. The ET-1+endothelin receptor type-A antagonist BQ-123 and the ETBR agonists sarafotoxin 6c and IRL-1620 caused less vasorelaxation and nitrate/nitrite production in RUPP than in Norm-Preg. The nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester reduced sarafotoxin 6c- and IRL-1620-induced relaxation in Norm-Preg but not in RUPP, supporting that ETBR-mediated nitric oxide pathway is compromised in RUPP. Reverse transcription polymerase chain reaction, Western blots, and immunohistochemistry revealed reduced endothelial ETBR expression in RUPP. Infusion of BQ-788 increased BP in Norm-Preg, and infusion of IRL-1620 reduced BP and ET-1 vasoconstriction and [Ca(2+)]i and enhanced ETBR-mediated vasorelaxation in RUPP. Thus, downregulation of microvascular vasodilator ETBR is a central mechanism in HTN-Preg, and increasing ETBR activity could be a target in managing preeclampsia.

Altered matrix metalloproteinase-2 and -9 expression/activity links placental ischemia and anti-angiogenic sFlt-1 to uteroplacental and vascular remodeling and collagen deposition in hypertensive pregnancy.

Preeclampsia is a complication of pregnancy manifested as maternal hypertension and often fetal growth restriction. Placental ischemia could be an initiating event, but the linking mechanisms leading to hypertension and growth restriction are unclear. We have shown an upregulation of matrix metalloproteinases (MMPs) during normal pregnancy (Norm-Preg). To test the role of MMPs in hypertensive-pregnancy (HTN-Preg), maternal and fetal parameters, MMPs expression, activity and distribution, and collagen and elastin content were measured in uterus, placenta and aorta of Norm-Preg rats and in rat model of reduced uteroplacental perfusion pressure (RUPP). Maternal blood pressure was higher, and uterine, placental and aortic weight, and the litter size and pup weight were less in RUPP than Norm-Preg rats. Western blots and gelatin zymography revealed decreases in amount and gelatinase activity of MMP-2 and MMP-9 in uterus, placenta and aorta of RUPP compared with Norm-Preg rats. Immunohistochemistry confirmed reduced MMPs in uterus, placenta and aortic media of RUPP rats. Collagen, but not elastin, was more abundant in uterus, placenta and aorta of RUPP than Norm-Preg rats. The anti-angiogenic factor soluble fms-like tyrosine kinase-1 (sFlt-1) decreased MMPs in uterus, placenta and aorta of Norm-Preg rats, and vascular endothelial growth factor (VEGF) reversed the decreases in MMPs in tissues of RUPP rats. Thus placental ischemia and anti-angiogenic sFlt-1 decrease uterine, placental and vascular MMP-2 and MMP-9, leading to increased uteroplacental and vascular collagen, and growth-restrictive remodeling in HTN-Preg. Angiogenic factors and MMP activators may reverse the decrease in MMPs and enhance growth-permissive remodeling in preeclampsia.

Combining two potential causes of metalloproteinase secretion causes abdominal aortic aneurysms in rats: a new experimental model.

Progress in understanding the pathophysiology of abdominal aortic aneurysms (AAA) is dependent in part on the development and application of effective animal models that recapitulate key aspects of the disease. The objective was to produce an experimental model of AAA in rats by combining two potential causes of metalloproteinase (MMP) secretion: inflammation and turbulent blood flow. Male Wistar rats were randomly divided in four groups: Injury, Stenosis, Aneurysm and Control (40/group). The Injury group received a traumatic injury to the external aortic wall. The Stenosis group received an extrinsic stenosis at a corresponding location. The Aneurysm group received both the injury and stenosis simultaneously, and the Control group received a sham operation. Animals were euthanized at days 1, 3, 7 and 15. Aorta and/or aneurysms were collected and the fragments were fixed for morphologic, immunohistochemistry and morphometric analyses or frozen for MMP assays. AAAs had developed by day 3 in 60-70% of the animals, reaching an aortic dilatation ratio of more than 300%, exhibiting intense wall remodelling initiated at the adventitia and characterized by an obvious inflammatory infiltrate, mesenchymal proliferation, neoangiogenesis, elastin degradation and collagen deposition. Immunohistochemistry and zymography studies displayed significantly increased expressions of MMP-2 and MMP-9 in aneurysm walls compared to other groups. The haemo-dynamic alterations caused by the stenosis may have provided additional contribution to the MMPs liberation. This new model illustrated that AAA can be multifactorial and confirmed the key roles of MMP-2 and MMP-9 in this dynamic remodelling process.

Giant right coronary artery aneurysm presenting as a paracardiac mass.

Coronary artery aneurysms, commonly atherosclerotic, are usually asymptomatic and can be diagnosed incidentally during investigation for ischemic heart disease or on autopsy. An asymptomatic giant right coronary artery aneurysm presenting as a paracardiac mass discovered on autopsy is reported here. Even though there was great concern due to its large size, complex atherosclerotic lesions, and potential obstruction of blood flow, it had no direct link to the cause of death. We also review the literature on giant right coronary artery aneurysms exceeding 5 cm in the last 10 years.