Comparisons of the release of vasodilator substances from left and right cardiac chambers of the isolated perfused rabbit heart: Implications for intraventricular thrombus formation

Prostacyclin (PgI(2)) and endothelium-derived nitric oxide (EDNO) are produced by the arterial and venous endothelium. In addition to their vasodilator action on vascular smooth muscle, both act together to inhibit platelet aggregation and promote platelet disaggregation. EDNO also inhibits platelet adhesion to the endothelium. EDNO and PgI(2) have been shown to be released from the cultured endocardial cells. In this study, we examined the release of vasoactive substances from the intact endocardium by using isolated rabbit hearts perfused with physiological salt solution (95% O(2)/5% CO(2), T = 37 degrees C). The right and left cardiac chambers were perfused through separate constant-flow perfusion loops (physiological salt solution, 8 ml min(-1)). Effluent from left and right cardiac, separately, was bioassayed on canine coronary artery smooth muscle, which had been contracted with prostaglandin F(2 alpha_)(2 x 10(-6) M) and no change in tension was exhibit. However, addition of calcium ionophore A23187 (10(-6) M) to the cardiac chambers` perfusion line induced vasodilation of the bioassay coronary ring, 61.4 +/- 7.4% versus 70.49 +/- 6.1% of initial prostaglandin F(2 alpha) contraction for the left and right cardiac chambers perfusate, respectively (mean +/- SEM, n = 10, p > 0.05). Production of vasodilator was blocked totally in the left heart but, only partially blocked in the right heart by adding indomethacin (10(-5) M) to the perfusate, respectively, 95.2 +/- 2.2% versus 41.5 +/- 4.8% (mean +/- SEM, n = 10, p < 0.05). 6-Keto prostaglandin F(1 alpha), measured in the endocardial superfusion effluent was also higher for the left cardiac chambers than for the right at the time of stimulation with the A23187, respectively, 25385.88 +/- 5495 pg/ml (n = 8) versus 13,132.45 +/- 1839.82 pg/ml (n = 8), (p < 0.05). These results showed that cyclooxygenase pathway plays major role in generating vasoactive substances for the left cardiac chamber endocardium; while it is not the main pathway for the right ventricular endocardium at which EDNO and PgI(2) Could act together and potentiate their antithrombogenic activities in isolated perfused rabbit heart. This may be an explanation for the intraventricular thrombus mostly seen in left ventricle rather than in right ventricle as a complication of myocardial infarction. (C) 2009 Elsevier Inc. All rights reserved.

Doxycycline Dose-dependently Inhibits MMP-2-Mediated Vascular Changes in 2K1C Hypertension

Hypertension induces vascular alterations that are associated with up-regulation of matrix metalloproteinases (MMPs). While these alterations may be blunted by doxycycline, a non-selective MMPs inhibitor, no previous study has examined the effects of different doses of doxycycline on these alterations. This is important because doxycycline has been used at sub-antimicrobial doses, and the use of lower doses may prevent the emergence of antibiotic-resistant microorganisms. We studied the effects of doxycycline at 3, 10 and 30 mg/kg per day on the vascular alterations found in the rat two kidneyone clip (2K1C) hypertension (n = 20 rats/group). Systolic blood pressure (SBP) was monitored during 4 weeks of treatment. We assessed endothelium-dependent and independent relaxations. Quantitative morphometry of structural changes in the aortic wall was studied, and aortic MMP-2 levels/proteolytic activity were determined by gelatin and in situ zymography, respectively. All treatments attenuated the increases in SBP in hypertensive rats (195.4 +/- 3.9 versus 177.2 +/- 6.2, 176.3 +/- 4.5, and 173 +/- 5.1 mmHg in 2K1C hypertensive rats treated with vehicle, or doxycycline at 3, 10, 30 mg/kg per day, respectively (all p < 0.01). However, only the highest dose prevented 2K1C-induced reduction in endothelium-dependent vasorelaxation (p < 0.05), vascular hypertrophy and increases in MMP-2 levels (all p < 0.05). In conclusion, our results suggest that relatively lower doses of doxycycline do not attenuate the vascular alterations found in the 2K1C hypertension model, and only the highest dose of doxycycline affects MMPs and vascular structure. Our results support the idea that the effects of doxycycline on MMP-2 and vascular structure are pressure independent.

Diabetes and Vascular Disease: Basic Concepts of Nitric Oxide Physiology, Endothelial Dysfunction, Oxidative Stress and Therapeutic Possibilities

The vascular manifestations associated with diabetes mellitus (DM) result from the dysfunction of several vascular physiology components mainly involving the endothelium, vascular smooth muscle and platelets. It is also known that hyperglycemia-induced oxidative stress plays a role in the development of this dysfunction. This review considers the basic physiology of the endothelium, especially related to the synthesis and function of nitric oxide. We also discuss the pathophysiology of vascular disease associated with DM. This includes the role of hyperglycemia in the induction of oxidative stress and the role of advanced glycation end-products. We also consider therapeutic strategies.

Chronic alcoholism associated with diabetes impairs erectile function in rats

OBJECTIVE To investigate the effects of chronic ethanol consumption and diabetes on nitric oxide (NO)-mediated relaxation of cavernosal smooth muscle (CSM). MATERIAL AND METHODS Male Wistar rats were divided into four groups: control, isocaloric, diabetic and ethanol-diabetic. The CSMs were mounted in organ chambers for measurement of isometric tension. Contraction of the strips was induced by electrical field stimulation (EFS, 1-32 Hz) and phenylephrine. We also evaluated the effect of ethanol consumption on the relaxation induced by acetylcholine (ACh; 0.01-1000 mu mol/L), sodium nitroprusside (SNP, 0.01-1000 mu mol/L) or EFS (1-32 Hz) in strips pre-contracted with phenylephrine (10 mu mol/L). Immunoexpression of endothelial NO synthase (eNOS) and inducible NOS (iNOS) was also accessed. RESULTS The endothelium-dependent relaxation induced by ACh was decreased in CSM from ethanol-diabetic rats when compared with the controls, with a mean (sem) of 21 (4) vs 37 (2)%. Similarly, the potency and maximal responses induced by SNP were reduced in the ethanol-diabetic [3.97 (0.38) and 85 (1)%, respectively] and diabetic groups [3.78 (0.56) and 81 (2)%, respectively] when compared with the controls [5.3 (0.22) and 90 (3)%, respectively] and isocaloric [5.3 (0.19) and 92 (1)%, respectively] groups. Noradrenergic nerve-mediated contractions of CSM in response to EFS were increased in rats from ethanol-diabetic and diabetic groups when compared with the control and isocaloric groups. Conversely, there were no differences in EFS-induced relaxation among the groups. The immunostaining assays showed overexpression of eNOS and iNOS in the CSM from diabetic and ethanol-diabetic rats when compared with the control and isocaloric rats. CONCLUSION There was an impairment of relaxation of CSM from ethanol-diabetic and diabetic rats that involved a decrease in the NO-cyclic guanosine monophosphate signalling pathway by endothelium-dependent mechanisms accompanied by a change in the CSM contractile sensitivity.

Co-existing squamous cell carcinoma and hemangioma on the ocular surface of a cat

A 14-year-old spayed female domestic short-haired cat was presented for evaluation of a mass in the right eye. Ophthalmic examination revealed a blind right eye and presence of two distinct masses: a pink and a red-to-brown mass, the latter occupying most of the cornea and part of the conjunctiva. Exenteration was performed under general anesthesia, and the ocular tissues were processed routinely for histopathology. Upon microscopic examination, a malignant epithelial neoplasm and a benign vascular neoplasm were present in the cornea. The conjunctiva and the third eyelid were also affected. Upon immunohistochemistry, the epithelial tumor was positive for cytokeratin and negative for vimentin and the endothelial tumor was negative for cytokeratin and positive for vimentin. A diagnosis of squamous cell carcinoma (SCC) and hemangioma was made. The SCC was affecting the cornea, bulbar conjunctiva (lateral and inferior) and the base of the third eyelid, whereas the hemangioma was affecting the cornea and medial limbus. To the authors` knowledge, this is the first report of concomitant SCC and hemangioma affecting the ocular surface in a cat.

Extramedullary plasmacytoma of the third eyelid gland in a dog

A case of extramedullary plasmacytoma of the third eyelid gland in a 7-year-old American Cocker Spaniel is reported. An enlargement of the third eyelid gland, abundant mucopurulent discharge, mild hyperemia and corneal pigmentation in the OD was present. Excisional biopsy of the mass revealed the gland was infiltrated and partially destroyed by a uniform population of neoplastic plasma cells. The neoplastic cells were positive for CD138, Ki-67 and lambda light chain. CD20, CD3, kappa light chain and cytokeratin were negative. Twelve months following surgery, no recurrence was observed. To the authors` knowledge, this is the first extramedullary plasmacytoma of the third eyelid gland reported in dogs.

Multiple imaging radiography at LNLS

An analyzer-based X-ray phase-contrast imaging (ABI) setup has been mounted at the Brazilian Synchrotron Light Laboratory (LNLS) for multiple imaging radiography (MIR) purposes. The algorithm employed for treating the MIR data collected at LNLS is described, and its reliability in extracting the distinct types of contrast that can be obtained with MIR is demonstrated by analyzing a test sample (thin polyamide wire). As a practical application, the possibility of studying ophthalmic tissues, corneal sequestra in this case, via MIR is investigated. (C) 2007 Elsevier B.V. All rights reserved.

Experimental infection with Rangelia vitalii in dogs: Acute phase, parasitemia, biological cycle, clinical-pathological aspects and treatment

Recently we conducted the molecular characterization of Rangelia vitalii, a protozoan with high pathogenicity for young dogs in southern Brazil. To date, the descriptions of the disease have been restricted to natural infection cases. Therefore, this study aimed to evaluate the parasitemia, biological cycles and clinical-pathological findings in dogs experimentally infected with R. vitalii in the acute phase of disease, and also aimed to test a therapeutic protocol based on the diminazene aceturate. For this study, we used 12 young dogs (females), separated into two groups. Group A was composed of healthy dogs, not-infected (n = 5), and Group B consisted of animals infected with R. vitalii (n = 7). After infection, the animals were monitored by blood smear examinations, which showed intra-erythrocytic forms of the parasite 5 days post-infection (PI). Parasitemia increased progressively in these animals and had the highest peak of circulating parasites between 9 and 11 days PI. Subsequently, the parasitemia reduced and the protozoan was seen inside the leukocytes in days 17, 19 and 21 PI. The most prominent clinical signs observed at the 20 day PI of experiment were lethargy, fever and anorexia. We observed a decrease of hematocrit of infected animals compared with not-infected dogs, featuring a moderate anemia. Pathological evaluation of one dog in Group B at day 21 PI revealed splenomegaly, hepatomegaly, lymphadenopathy, and hemorrhages at necropsy. Histological examination showed only follicular hyperplasia in the spleen and lymph nodes, and the etiologic agent in the vascular endothelium. At 21 days PI, it was performed the treatment of dogs in Group B (n = 6) with a single dose of diminazene aceturate, which showed a curative efficacy of 100% in cleaning R. vitalii from blood of infected dogs. (C) 2011 Elsevier Inc. All rights reserved.

Lp95, a novel leptospiral protein that binds extracellular matrix components and activates e-selectin on endothelial cells

Objectives: The study of a predicted outer membrane leptospiral protein encoded by the gene LIC12690 in mediating the adhesion process. Methods: The gene was cloned and expressed in Escherichia coli BL21 (SI) strain by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and used to assess its ability to activate human umbilical vein endothelial cells (HUVECs). Results: The recombinant leptospiral protein of 95 kDa, named Lp95, activated E-selectin in a dose-dependent fashion but not the intercellular adhesion molecule 1 (ICAM-1). In addition, we show that pathogenic and non-pathogenic Leptospira are both capable to stimulate endothelium E-selectin and ICAM-1, but the pathogenic L. interrogans serovar Copenhageni strain promotes a statistically significant higher activation than the non-pathogenic L. biflexa serovar Patoc (P < 0.01). The Lp95 was identified in vivo in the renal tubules of animal during experimental infection with L. interrogans. The whole Lp95 as well as its fragments, the C-terminal containing the domain of unknown function (DUF), the N-terminal and the central overlap regions bind laminin and fibronectin ECM molecules, being the binding stronger with the DUF containing fragment. Conclusion: This is the first leptospiral protein capable to mediate the adhesion to ECM components and the activation of HUVECS, thus suggesting its participation in the pathogenesis of Leptospira. (C) 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Placentation in the Amazonian manatee (Trichechus inunguis)

Evidence from several sources supports a close phylogenetic relationship between elephants and sirenians. To explore whether this was reflected in similar placentation, we examined eight delivered placentae from the Amazonian manatee using light microscopy and immunohistochemistry. In addition, the fetal placental circulation was described by scanning electron microscopy of vessel casts. The manatee placenta was zonary and endotheliochorial, like that of the elephant. The interhaemal barrier comprised maternal endothelium, cytotrophoblasts and fetal endothelium. We found columnar trophoblast beneath the chorionic plate and lining lacunae in this region, but there was no trace in the term placenta of haemophagous activity. The gross anatomy of the cord and fetal membranes was consistent with previous descriptions and included a four-chambered allantoic sac, as also found in the elephant and other afrotherians. Connective tissue septae descended from the chorionic plate and carried blood vessels to the labyrinth, where they gave rise to a dense capillary network. This appeared to drain into shorter vessels near the chorionic plate. The maternal vasculature could not be examined in the same detail, but maternal capillaries ran rather straight and roughly parallel to the fetal ones. Overall, there is a close resemblance in placentation between the manatee and the elephant.

Cystic hygroma: characterization by computerized tomography

Lymphangiomas are benign nonencapsulated lesions composed of sequestered noncommunicating lymphoid tissue lined by lymphatic endothelium and are thought to be caused by congenital obstruction of lymphatic drainage. They are subclassified by vessel size, such as the capillary, which is rare and located in subcutaneous tissue, cavernous (located about the mouth and tongue), and cystic (cystic hygromas). The cystic hygromas show a predilection for the neck (75%) and maxilla (20%), and the remaining 5% arise in rare locations such as the mediastinum, retroperitoneum, bone, kidney, colon, liver, spleen and scrotum. Only 3%-10% of neck lesions extend into the mediastinum. In this paper, we report a rare case of cystic hygroma with a huge dimension discussing the use of computed tomography scanning for diagnosis.

Characterization of a Local Renin-Angiotensin System in Rat Gingival Tissue

Background: The systemic renin-angiotensin system (RAS) promotes the plasmatic production of angiotensin (Ang) II, which acts through interaction with specific receptors. There is growing evidence that local systems in various tissues and organs are capable of generating angiotensins independently of circulating RAS. The aims of this study were to investigate the expression and localization of RAS components in rat gingival tissue and evaluate the in vitro production of Ang II and other peptides catalyzed by rat gingival tissue homogenates incubated with different Ang II precursors. Methods: Reverse transcription - polymerase chain reaction assessed mRNA expression. Immunohistochemical analysis aimed to detect and localize renin. A standardized fluorimetric method with tripeptide hippuryl-histidyl-leucine was used to measure tissue angiotensin-converting enzyme (ACE) activity, whereas high performance liquid chromatography showed products formed after the incubation of tissue homogenates with Ang I or tetradecapeptide renin substrate (TDP). Results: mRNA for renin, angiotensinogen, ACE, and Ang II receptors (AT(1a), AT(1b), and AT(2)) was detected in gingival tissue; cultured gingival fibroblasts expressed renin, angiotensinogen, and AT(1a) receptor. Renin was present in the vascular endothelium and was intensely expressed in the epithelial basal layer of periodontally affected gingival tissue. ACE activity was detected (4.95 +/- 0.89 nmol histidyl-leucine/g/minute). When Ang I was used as substrate, Ang 1-9 (0.576 +/- 0.128 nmol/mg/minute), Ang II (0.066 +/- 0.008 nmol/mg/minute), and Ang 1-7 (0.111 +/- 0.017 nmol/mg/minute) were formed, whereas these same peptides (0.139 +/- 0.031, 0.206 +/- 0.046, and 0.039 +/- 0.007 nmol/mg/minute, respectively) and Ang 1 (0.973 +/- 0.139 nmol/mg/minute) were formed when TDP was the substrate. Conclusion: Local RAS exists in rat gingival tissue and is capable of generating Ang II and other vasoactive peptides in vitro. J Periodontol 2009;80:130-139.

Pyrrolidine dithiocarbamate down-regulates vascular matrix metalloproteinases and ameliorates vascular dysfunction and remodelling in renovascular hypertension

BACKGROUND AND PURPOSE Mounting evidence implicates matrix metalloproteinase (MMP) in the vascular dysfunction and remodelling associated with hypertension. We tested the hypothesis that treatment with pyrrolidine dithiocarbamate (PDTC), which interferes with NF-kappa B-induced MMPs gene transcription, could exert antihypertensive effects, prevent MMP-2 and MMP-9 up-regulation, and protect against the functional alterations and vascular remodelling of two-kidney, one clip (2K1C) hypertension. EXPERIMENTAL APPROACH Sham-operated or hypertensive rats were treated with vehicle or PDTC (100 mg.Kg(-1).day(-1)) by gavage for 8 weeks. Systolic blood pressure (SBP) was monitored weekly. Aortic rings were isolated to assess endothelium-dependent relaxations. Quantitative morphometry of structural alterations of the aortic wall was carried out in haematoxylin/eosin sections. Formation of vascular reactive oxygen species (ROS), and inducible (i) NOS and phosphorylated-p65 NF-kappa B subunit expression were measured in the aortas. MMP-2 and MMP-9 aortic levels and gelatinolytic activity were determined by gelatin and in situ zymography and by immunofluorescence. KEY RESULTS Treatment with PDTC attenuated the increases in SBP and prevented the endothelial dysfunction associated with 2K1C hypertension. Moreover, PDTC reversed the vascular aortic remodelling, the increases in aortic ROS levels and in iNOS and phosphorylated-p65 NF-kappa B expression found in 2K1C rats. These effects were associated with attenuation of 2K1C up-regulation of aortic MMP-2 and MMP-9 levels and gelatinolytic activity. CONCLUSION AND IMPLICATIONS These findings suggest that PDTC down-regulates vascular MMPs and ameliorates vascular dysfunction and remodelling in renovascular hypertension, thus providing evidence supporting the suggestion that PDTC is probably a good candidate to be used to treat hypertension.

Spironolactone and hydrochlorothiazide exert antioxidant effects and reduce vascular matrix metalloproteinase-2 activity and expression in a model of renovascular hypertension

Background and purpose: Increased oxidative stress and up-regulation of matrix metalloproteinases (MMPs) may cause structural and functional vascular changes in renovascular hypertension. We examined whether treatment with spironolactone (SPRL), hydrochlorothiazide (HCTZ) or both drugs together modified hypertension-induced changes in arterial blood pressure, aortic remodelling, vascular reactivity, oxidative stress and MMP levels and activity, in a model of renovascular hypertension. Experimental approach: We used the two-kidney,one-clip (2K1C) model of hypertension in Wistar rats. Sham-operated or hypertensive rats were treated with vehicle, SPRL (25 mg center dot kg-1 center dot day-1), HCTZ (20 mg center dot kg-1 center dot day-1) or a combination for 8 weeks. Systolic blood pressure was monitored weekly. Aortic rings were isolated to assess endothelium-dependent and -independent relaxations. Morphometry of the vascular wall was carried out in sections of aorta. Aortic NADPH oxidase activity and superoxide production were evaluated. Formation of reactive oxygen species was measured in plasma as thiobarbituric acid-reactive substances. Aortic MMP-2 levels and activity were determined by gelatin and in situ zymography, fluorimetry and immunohistochemistry. Key results: Treatment with SPRL, HCTZ or the combination attenuated 2K1C-induced hypertension, and reversed the endothelial dysfunction in 2K1C rats. Both drugs or the combination reversed vascular aortic remodelling induced by hypertension, attenuated hypertension-induced increases in oxidative stress and reduced MMP-2 levels and activity. Conclusions and implications: SPRL or HCTZ, alone or combined, exerted antioxidant effects, and decreased renovascular hypertension-induced MMP-2 up-regulation, thus improving the vascular dysfunction and remodelling found in this model of hypertension.

Evidence for the involvement of matrix metalloproteinases in the cardiovascular effects produced by nicotine

Nicotine plays a role in smoking-associated cardiovascular diseases, and may upregulate matrix metalloproteinase (MMP)-2 and MMP-9. We examined whether nicotine induces the release of MMP-2 and MMP-9 by rat smooth muscle cells (SMC), and whether doxycycline (non-selective MMP inhibitor) inhibits the vascular effects produced by nicotine. SMC were incubated with nicotine 0, 50, and 150 nM for 48 h. MMP-2 and MMP-9 levels in the cell supernatants were determined by gelatin zymography. The acute changes in mean arterial pressure caused by nicotine 2 mu mol/kg (or saline) were assessed in rats pretreated with doxycycline (or saline). We also examined whether doxcycline (30 mg/Kg, i.p., daily) modifies the effects of nicotine (10 mg/kg/day; 4 weeks) on the endothelium-dependent relaxations of rat aortic rings. Aortic MMP-2 levels were assessed by gelatin zymography. Aortic gelatinolytic activity was assessed using a gelatinolytic activity kit. MMP-2 and MMP-9 levels increased in the supernatant of SMC cells incubated with nicotine 150 nM (P<0.05) but not with 50 nM. Nicotine (2 mu mol/kg) produced lower increases in the mean arterial pressure in rats pretreated with doxycycline than those found in rats pretreated with saline (26 +/- 4 vs. 37 +/- 4 mmHg, respectively; P<0.05). Nicotine impaired of the endothelium-dependent responses to acetylcholine, and treatment with doxycycline increased the potency (pD2) by approximately 25% (P<0.05). While we found no significant differences in aortic MMP-2 levels, nicotine significantly increased gelatinolytic activity (P<0.05). These findings suggest that nicotine produces cardiovascular effects involving MMPs. It is possible that MMPs inhibition may counteract the effects produced by nicotine. (C) 2009 Elsevier B.V. All rights reserved.