Ultrastructural Study of Platelet Behavior and Interrelationship in Sprouting and Intussusceptive Angiogenesis during Arterial Intimal Thickening Formation.

Platelets in atherosclerosis, bypass stenosis, and restenosis have been extensively assessed. However, a sequential ultrastructural study of platelets in angiogenesis during the early phases of these lesions has received less attention. Our objective was the study of platelets in angiogenesis and vessel regression during intimal thickening (IT) formation, a precursor process of these occlusive vascular diseases. For this purpose, we used an experimental model of rat occluded arteries and procedures for ultrastructural observation. The results show (a) the absence of platelet adhesion in the de-endothelialized occluded arterial segment isolated from the circulation, (b) that intraarterial myriad platelets contributed from neovessels originated by sprouting angiogenesis from the periarterial microvasculature, (c) the association of platelets with blood components (fibrin, neutrophils, macrophages, and eosinophils) and non-polarized endothelial cells (ECs) forming aggregates (spheroids) in the arterial lumen, (d) the establishment of peg-and-socket junctions between platelets and polarized Ecs during intussusceptive angiogenesis originated from the EC aggregates, with the initial formation of IT, and (e) the aggregation of platelets in regressing neovessels ('transitory paracrine organoid') and IT increases. In conclusion, in sprouting and intussusceptive angiogenesis and vessel regression during IT formation, we contribute sequential ultrastructural findings on platelet behavior and relationships, which can be the basis for further studies using other procedures.

Cross talk between TGF beta and TNF alfa in regression of myointimal hyperplasia.

BACKGROUND: The phenomena involved in regression of arterial myointimal hyperplasia have not been analyzed in detail. MATERIALS AND METHODS: In 24 Lewis rats, a 1-cm-long venous graft, obtained from syngenic Lewis rats, was implanted in the infrarenal aorta. After 4 wk, the grafts were removed and analyzed using scanning electron microscopy and histochemistry. The grafts showed evidence of myointimal hyperplasia; 16 of these explanted grafts were reimplanted in the vein circulation of syngenic Lewis rats. These grafts were harvested 2 wk (8 animals) and 8 wk (8 animals) later, showing complete regression of myointimal hyperplasia. RESULTS: Regression of experimental myointimal hyperplasia was correlated with the simultaneous and complementary action of Transforming Growth Factor beta and Tumor Necrosis Factor alfa. Inflammatory cytokines (IL1, IL2, and IL6) inhibit Tumor Necrosis Factor alfa-induced apoptosis. CONCLUSIONS: Regression of myointimal hyperplasia is an active process, which implies the action of several inhibitory factors. The analysis of these phenomena can lead to new therapeutic approaches to prevent myointimal hyperplasia progression.

Bioinspired One-Dimensional Nano-Wrinkles Guide Liquid Behaviors at the Liquid-Solid Interfaces.

Learning from nature concerning how nanostructured surfaces interact with liquids may provide insight into better understanding of inside living biological interfaces bearing these nanostructures and further development of innovative materials contacting water. Here we investigate the dynamic behaviour of water droplet interacting with one-dimensional nano-wrinkles of different size on polydimethylsiloxane (PDMS) surface. The structure design of the variationally one-dimensional nano-wrinkles is inspired by in vivo responding topographic changes in aortic intima, which was characterized with liquid-phase atomic force microscopy. We show here that increasing the amplitude of the wrinkles promotes the spreading and energy dissipation of liquid droplets on the wrinkled interfaces. This result suggests a possible bio-protection mechanism of blood vessels via its structural changes on the aortic intima against elevated flowing blood, and provides a basis for tuning interfacial nanostructure of optimal durability against wearing by the liquid behaviors.

Internal mammary artery atherosclerosis: an ultrastructural study of two cases.

Atherosclerosis of the internal mammary artery (IMA) is generally regarded as a rare (but existent) pathological entity with only a few cases reported in the most recent literature. The only study which to our knowledge has investigated the ultrastructural features of IMA atherosclerosis, demonstrate the presence of endothelial cells loss, defects of internal elastic lamina with no evidence of lipid accumulation. In the present study, we describe two cases of IMA atherosclerosis in which ultrastructural analysis revealed the presence of a typical atherosclerotic plaque morphology with infiltration of inflammatory cells, formation of intraplaque lipid pools, and accumulation of lipid-laden foam cells throughout the thickened intima, never described in this rare lesion before. Microscopically, the lesions were also characterized by intimal thickening, invagination of endothelial cells, migration of smooth muscle cells with splitting, fenestration and/or fragmentation of the elastic sheets. Our observations add new data to the scarce and contradictory literature and to this largely understudied vascular disorder.

Changes of lysosomes in the earliest stages of the development of atherosclerosis.

One of hypotheses of atherosclerosis is based on a presumption that the zones prone to the development of atherosclerosis contain lysosomes which are characterized by enzyme deficiency and thus, are unable to dispose of lipoproteins. The present study was undertaken to investigate the characteristics and changes of lysosomes in the earliest stages of the development of atherosclerosis. Electron microscopic immunocytochemistry revealed that there were certain changes in the distribution of CD68 antigen in lysosomes along the 'normal intima-initial lesion-fatty streak' sequence. There were no significant changes found in the key mRNAs encoding for the components of endosome/lysosome compartment in initial atherosclerotic lesions, but in fatty streaks, the contents of EEA1 and Rab5a mRNAs were found to be diminished while the contents of CD68 and p62 mRNAs were increased, compared with the intact tissue. The study reinforces a view that changes occurring in lysosomes play a role in atherogenesis from the very earlier stages of the disease.

Increased shedding of microvesicles from intimal smooth muscle cells in athero-prone areas of the human aorta: implications for understanding of the predisease stage.

OBJECTIVE: This study evaluated whether a change in the content of matrix microvesicles might occur at the preatherosclerotic stage. METHODS: Applying quantitative electron microscopic and immunohistochemical analyses, two areas of grossly normal segments of the thoracic aorta were compared: atherosclerosis-prone (AP) areas, situated at the dorsal aspect of the aorta along the rows of intercostal branch origins, and atherosclerosis-resistant (AR) areas, situated at the corresponding sites of the ventral aspect of the aorta. RESULTS: The electron microscopic analysis showed that there were 1.4 times more microvesicles in AP areas than AR areas (p = 0.019). It was found that matrix microvesicles originated as a result of blebbing and shedding of surface membranes of smooth muscle cells. A quantitative analysis of the expression of ADP-ribosylation factor 6 (ARF6), which is known to be involved in membrane trafficking and microvesicle formation, showed that ARF6 expression was 1.3 times higher in AP areas than that in AR areas (p = 0.006). There was a positive correlation between the content of matrix microparticles and the expression of ARF6 by intimal smooth muscle cells (r = 0.61; p < 0.0001). CONCLUSION: The present study supports the concept that alterations of the arterial intima occur at the predisease stage.

Ultrastructural features of human atherosclerosis.

Healthy human arteries are composed of three layers: the intima, the media, and the adventitia. Endothelial cells, which form the tunica intima, provide the physical interface between blood and surrounding tissue, regulate nutrient and blood component traffic, and participate in many physiologic events, such as hemostasis, inflammation, and angiogenesis. Within the tunica media, smooth muscle cells and extracellular matrix proteins, such as elastin, collagen, and proteoglycans, are quantitatively the largest components of the aortic vascular wall. The structural changes with atherosclerosis are currently considered degenerative phenomena, which primarily involve a sequence of reactions within the intima and include monocyte recruitment and macrophage formation, lipid deposition, smooth muscle cell migration, proliferation, and extracellular matrix synthesis. The molecular and cellular mechanisms underlying the disease cascade have been thoroughly investigated in experimental animals and cell culture, but the question of how these models can correctly mimic the human course of the disease remains open to debate. In the present review the basic structure of healthy human arteries and the pathological events occurring during the atherosclerotic process have been examined by both transmission and scanning electron microscopy. Human atherosclerotic lesions are presented and described in the following order: initial lesions, fatty dots and streaks, intermediate lesions, atheroma and fibrofatty plaques, and complicated lesions.

[Hepatic structure remodeling after an experimental pulmonary trunk stenosis and following its operative correction].

Changes in the liver were studied in 25 puppies with experimental pulmonary trunk stenosis of 6-12 months duration and 10 animals after the elimination of this defect. Control group included 10 dogs of the corresponding age. A complex of histological, morphometric, electron microscopic and immunohistochemical methods was used. During the modeling of pulmonary trunk stenosis, the resistance of hepatic afferent vessels to hepatic blood flow was increased due to the venous-arterial and venous-venous reactions. In the arteries, the bundles of smooth myocytes (SM) of the intimal muscle were formed together with the musculo-elastic sphincters, polypoid cushions, while in the branches of the portal vein, the intimal SM bundles and the valves appeared. In the efferent veins, the muscular elevations were hypertrophied. In all the vessels the thickening of the walls was observed, and in the media of the arteries, there were signs of sclerosis and the increased expression of alpha-smooth muscle actin (alpha-SMA). Hepatocytes demonstrated marked ultrastructural changes: mitochondrial matrix swelling, partial destructions of their cristae, dilation of endoplasmic reticulum cisterns. After the elimination of the defect, previously formed vascular adaptation reactions were found to disappear, the tone of the blood vessels in the liver decreased, causing the regression of hypertrophic changes of their media. The number of the arterial blood vessels with intimal muscle, sphincters and cushions decreased. The expression of alpha-SMA in the media of the arteries was also reduced. In hepatic efferent veins, the muscular elevations became attenuated. The dystrophic changes in hepatocytes regressed at both light-microscopic and the ultrastructural level.

Implication of vitamin A deficiency on vascular injury related to inflammation and oxidative stress. Effects on the ultrastructure of rat aorta.

BACKGROUND: Vitamin A deficiency induces activation of NF-kB and impairs activities of antioxidant enzymes in aorta. AIM OF THE STUDY: We study the effect of vitamin A deficiency on the aorta histoarchitecture and the possibly contribution of its prooxidant and inflammatory effects to artery alterations. METHODS: Twenty-one-day-old Wistar male rats were fed during 3 months with vitamin A-deficient diet (-A, n = 8) or the same diet containing 8 mg of retinol palmitate/kg of diet (+A, control, n = 8). In aortas, thiobarbituric reactive substances and reduced glutathione levels were measured by spectrophotometry. Expressions of TNF-alpha, NOX-2, VCAM-1, and TGF-beta1 were assessed by RT-PCR and Western Blot. The morphology of aorta was examined by light and transmission electron microscopy. RESULTS: In -A rats, high levels of TBARS in serum and aorta and low levels of GSH in aorta were found. An increased expression of TNF-alpha, NOX-2, VCAM-1, and TGF-beta1 in aorta from -A rats was observed. Examination of the intimal layer by light microscopy indicated the presence of an irregular surface in -A aortas. TEM studies showed large vacuoles and multivesicular bodies along the endothelium and also multivesicular bodies in the subendothelial space of aortas from -A rats. Furthermore, the histological appearance of internal elastic lamina was different from control. Small vesicles in the medial layer were observed in aortas from vitamin A-deficient rats. CONCLUSIONS: Vitamin A deficiency produces histoarchitectural alterations in aorta, which can be associated, at least in part, to the oxidative stress and inflammation induced by vitamin A deficiency.

Paradoxical effects of streptozotocin-induced diabetes on endothelial dysfunction in stroke-prone spontaneously hypertensive rats.

Although both diabetes and hypertension are risk factors for cardiovascular disease, the role of hyperglycaemia per se in endothelial dysfunction is controversial. This study was designed to examine whether hyperglycaemia, or streptozotocin-induced diabetes, could aggravate endothelial dysfunction in stroke-prone spontaneously hypertensive rats (SHRSP). Hyperglycaemia was induced by streptozotocin in 2-month-old SHRSP and age-matched normotensive Wistar-Kyoto (WKY) rats. The aorta was isolated 8 weeks after induction of hyperglycaemia to record its function and to examine its morphology with transmission electron microscopy. Endothelial/inducible nitric oxide synthase (eNOS/iNOS) and inducible/constitutive haem oxygenase (HO-1/HO-2) levels were determined with Western blotting. Aortic endothelial function and production of reactive oxygen species and nitric oxide were assayed after incubation in vitro in hyperglycaemic, hyperosmolar solution. Streptozotocin-induced diabetes of 8 weeks duration did not result in endothelial dysfunction in normotensive WKY rats. In contrast, hyperglycaemic WKY rats showed significantly enhanced endothelium-dependent vasodilatation, which was abrogated by simultaneous blocking of NOS and HO. The enhanced vasodilatation was associated with elevation of vascular eNOS and HO-1. Significant endothelial dysfunction and massive macrophage-monocyte infiltration were found in SHRSP aorta (the ratio of the number of macrophages to endothelial cells in the intima, expressed as a percentage, was 20.9 ± 2.8% in SHRSP versus 1.9 ± 0.5% in WKY rats, P < 0.01), which was attenuated significantly in hyperglycaemic SHRSP (11.3 ± 1.6%, P < 0.01 versus SHRSP). Acute hyperglycaemia (10 min) aggravated endothelial dysfunction in SHRSP, with a marked increase in intracellular reactive oxygen species and NO production. Sustained in vitro incubation in hyperglycaemic/hyperosmolar conditions (addition of an extra 50 mmol L(-1) of glucose or mannitol to the usual buffer, to produce a final osmolarity of 350 mosmol L(-1)) for 5 h enhanced endothelium-dependent vasodilatation, with elevated vessel NO production and upregulation of eNOS/HO-1 proteins. Sustained hyperglycaemia does not aggravate endothelial dysfunction and macrophage infiltration in SHRSP. Hyperglycaemia/hyperosmolarity-induced upregulation of eNOS and HO-1 may play a role in this paradoxical adaptation of endothelial function.

The effect of L-arginine or L-citrulline supplementation on biochemical parameters and the vascular aortic wall in high-fat and high-cholesterol-fed rats.

The aim of the present study is to investigate the potential role of L-arginine or L-citrulline in rats fed high-fat and high-cholesterol (HFC) diet. HFC feeding increased significantly serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, urea and all lipid profiles and decreased significantly serum high-density lipoprotein-cholesterol (HDL-c) and non significantly serum nitric oxide levels. L-arginine or L-citrulline administration reversed the increase in serum AST and ALT activities, urea and all lipid profiles. These effects were associated with a concomitant increase in HDL-c and nitric oxide levels. In general, rats fed HFC diet and orally treated with L-arginine or L-citrulline had higher relative percentage of 18:0, 20:0 and 22:6 and lower 16:0 fatty acids than rats fed HFC diet. Light and transmission electron microscopic findings of the thoracic aorta confirmed the biochemical results and demonstrated structural changes in the endothelial cells of the intimal layer, medial smooth muscle cells as well as in the adventitial layer in HFC fed-animals. However, these findings indicate little structural alterations in animals supplemented with L-arginine or L-citrulline along with HFC feeding. In the present study, L-arginine or L-citrulline was effective hypocholesterolemic and hypolipidemic agents in rats.

Role of echocardiographic left ventricular mass and carotid intima-media thickness in the cardiovascular risk assessment of asymptomatic patients with type 2 diabetes mellitus.

BACKGROUND: Standard cardiovascular (CV) risk assessment may underestimate risk in people with type 2 diabetes mellitus (T2DM). Cardiac and vascular imaging to detect subclinical disease may augment risk prediction. This study investigated the association between CV risk, left ventricular hypertrophy (LVH) and carotid intima-media thickness (CIMT) in patients with T2DM free of CV symptoms. METHODS: People with T2DM without known CV disease were recruited from general practice. The 5-year risk of CV events was calculated using an adjusted Framingham equation and the prevalence of LVH and abnormal CIMT across bands of CV risk assessed. In those at intermediate risk, the number needed to scan (NNS) to reclassify one person to high risk was calculated across the group and compared in those above and below 55 years. The association between LV mass and CIMT was also assessed. RESULTS: Mean age 57 years (SD11), 51% female. Median 5-year CV risk 14.3% (interquartile range 10.3, 19.5), 51% had LVH (American Society of Echocardiography criteria) and 31% an abnormal CIMT (age and sex criteria). In the 52% at intermediate risk, 37% had LVH and 36% an abnormal CIMT. The NNS was 1.7 using both imaging techniques, 2.7 using cardiac imaging alone or 2.8 using vascular imaging alone. Almost twice as many people >55 years had an abnormal CIMT than those <55 years. CONCLUSIONS: Cardiac and vascular imaging to detect subclinical disease can be used to augment prediction of CV risk in people with T2DM at intermediate risk. The value of reclassifying risk is as yet unproven and requires outcome data from intervention studies.

Scanning and transmission electron microscopic observation of femoral head feeding vessels in stroke-prone spontaneously hypertensive rats.

Stroke-prone spontaneously hypertensive rats (SHRSP) are known to show necrosis of the femoral head with a frequency of about 50%. This rat has thus been used as an animal model for necrosis of the femoral head in many studies. In a detailed investigation of feeding vessel disorders that cause femoral head necrosis, we observed changes over time in the feeding vessels using scanning electron microscopy and transmission electron microscopy. In scanning electron microscopy of vascular casts, abnormal findings in feeding vessels of SHRSP with aging from the immature stage included contortion and bending in the lumen with overall narrowing. Under transmission electron microscopy, decreased numbers of smooth muscle cells and increased amounts of collagen fibers were marked, and these changes with hypertrophy of vascular walls might be similar to those of arteriolosclerosis. The structural changes first revealed by transmission electron microscopic observation might cause the friability of the feeding vessels so that contortion and bending occurred, suggesting transient obstruction of blood flow to the femoral head and subsequent induction of femoral head necrosis. These findings should help in understanding the causes of femoral head necrosis in humans, including Perthes' disease.

Rapamycin-eluting stents in the arterial duct: experimental observations in the pig model.

BACKGROUND: Maintaining arterial duct patency by stent implantation may be advantageous in congenital heart disease management algorithms. Rapamycin, an immunosuppressant drug that demonstrates antiproliferative properties and inhibits smooth muscle cell migration, may deter the intimal hyperplasia that occurs during spontaneous closure and after-stent implantation of the arterial duct. METHODS AND RESULTS: Twenty-eight Yorkshire piglets (7 to 11 days old; weight, 2.2 to 4.9 kg) underwent stent implantation of the arterial duct (rapamycin-eluting (n=14) or bare metal (n=14) stents, 3.5-mm diameter) and were euthanized at 2, 4, and 6 weeks. Dissected arterial ducts were analyzed for lumen diameter, smooth muscle cell, and extracellular matrix components. Isolated arterial duct-derived smooth muscle cells were cultured in the presence or absence of rapamycin. Cellular proliferation rates were assessed by Ki-67 detection and [(3)H]-thymidine incorporation. No significant neointimal proliferation was present in either stent type at 2 weeks. At 4 weeks, the median luminal diameters of the bare metal stents were 87% (P=0.009), 54% (P=0.004), and 77% (P=0.004) that of the drug-eluting stents at the middle and aortic and pulmonary artery ends, respectively. At 6 weeks, the median luminal diameters of the bare metal stents were 0% (P=0.18), 5% (P=0.25), and 61% (P=0.13) that of the drug-eluting stents at the same respective levels. Complete histological occlusion was found in at least 1 level of the lumen in 9 pigs: 1 (17%) in the BMS group at 4 weeks, 5 (83%) in the BMS group at 6 weeks, and 3 (50%) in the DES group at 6 weeks. In vitro studies demonstrated 50%-lower proliferation rates in rapamycin-treated cultures of duct-derived smooth muscle cell cultures (P<0.001). CONCLUSIONS: Rapamycin has antiproliferative actions on the arterial duct. Drug-eluting stents may be a more efficient tool than current palliative options for maintaining patency in critically duct-dependent states, but there may be a finite time-related benefit.

Dose-dependent ultrastructural and morphometric alterations after erythropoietin treatment in rat femoral artery vasospasm model.

PURPOSE: Cerebral vasospasm is the common cause of poor outcome after aneurysmal subarachnoid hemorrhage (aSAH). Although many agents are experimentally and clinicaly used to protect or recover from vasospasm, an effective neurotherapeutic drug is still missing. Erythropoietin (EPO) is recently a promising candidate. The aim of this study is to investigate the dose-dependent effects of recombinant human EPO (rhEPO) on arterial wall in a rat femoral artery vasospasm model. METHODS: Thirty two animals were divided into four groups: vasospasm without any treatment (group A), vasospasm +250 IU/kg rhEPO group (group B), vasospasm +500 IU/kg rhEPO group (group C), and control group (group D). Rat femoral artery vasospasm model was used. For groups B and C, 7 days of 250 IU/kg and 500 IU/kg intraperitoneal rhEPO in 0.3 ml saline were administered respectively; and for groups A and D, 0.3 ml saline were administered intraperitoneally without any treatment. After 7 days, histological and morphometric analyses were carried out. RESULTS: Vasospasm alone group demonstrated the highest vessel wall thicknesses, comparing to other groups (p < 0.001). While for groups B and C, vessel wall thickness values were significantly higher than the control group (p < 0.001), between these two groups, there was no significant difference achieved (p > 0.05). CONCLUSION: In our study, there was no significant difference between the two rhEPO treatment groups, but rhEPO treatment was shown to be histologically and morphometrically effective in vasospasm. However, if dosage of EPO treatment is augmented, successful results may be achieved.

Morphological changes in varicocele veins: ultrastructural study.

BACKGROUND: Varicocele consists of dilatation of the pampiniform venous plexus and the internal spermatic veins. It is present in 15% of male population and is a common cause of male infertility. OBJECTIVE: To describe the normal structure of the internal spermatic vein and the morphological changes in grade 3 varicocele. METHODS: The authors dissected and analyzed a 2- to 3-cm tract of the pampiniform venous plexus of 20 patients undergoing varicocelectomy for left varicocele and of 10 consecutive patients undergoing surgery for left inguinal hernia. The histological examination was performed with hematoxylin­eosin and Masson trichrome stains. The ultrastructural evaluation was done using both scanning and transmission electron microscopy. RESULTS: Compared with normal internal spermatic veins, varicocele veins showed narrowing and/or obliteration of the lumens, destruction of the endothelial cells, invagination of the intima, and deposition of collagen bundles in the media (light microscopy). The ultrastructural changes in varicocele veins included elongation of the endothelial cells with features of cellular damage, loss of the internal elastic lamina, and the appearance of ghost bodies and degenerative vacuoles in the subendothelial layer. CONCLUSIONS: The authors believe this is the first report analyzing ultrastructual changes in normal human internal spermatic vein samples and in varicocele. The underlying molecular mechanisms of these changes await further studies.

Structural organisation of tunica intima in the aorta of the goat.

The structural organisation of tunica intima in the aorta is important for its integrity, prediction, and diagnosis of atherosclerosis. The goat is a suitable model for cardiovascular studies, but the structure of its tunica intima is scarcely reported. This study, therefore, aimed to describe features of the goat aortic tunica intima by light and transmission electron microscopy. Sixteen healthy male domestic goats (capra hircus) aged between 6 and 24 months were used: 8 for light and 8 for electron microscopy. The animals were euthanised with sodium pentabarbitone 20 mg/mL and fixed with 3% phosphate buffered glutaraldehyde. For light microscopy, specimens from various regions of the aorta were routinely processed for paraffin embedding and 7 mm sections stained with Mason's trichrome. Those for transmission electron microscopy were post fixed in osmium tetroxide, embedded in Durcupan, and ultrathin sections stained with uranyl acetate and counter stained with lead citrate. Endothelium comprises round and squamous cells, linked to the subendothelial material by a simple and sometimes lamellated basement membrane. In the subendothelial zone, a heterogenous population of cells are connected with interlinked collagen and elastic fibres. Both cells and fibres are connected to the internal elastic lamina. The composite structure and interlinkages in the tunica intima permit unitary function and increase mechanical strength, thus enabling it to withstand haemodynamic stress.

[Effect of sulodexide on aortic vasodilation capacity and associated morphological changes in rats with streptozotocin-induced diabetes]. / Efecto del sulodexide sobre la capacidad de relajación y alteraciones estructurales de la arteria aorta en ratas diabéticas por estreptozotocina.

Endothelial dysfunction (ED) is observed in patients with hypercholesterolemia, arterial hypertension, obesity and diabetes mellitus. Recent evidences suggest the involvement of glycosaminoglycans (GSG) in ED. We evaluated the effect of sulodexide (SLD), a natural GSG used in albuminuria and ischemic diabetes treatment, on arterial relaxation and vascular morphological changes in a diabetic type I model. Diabetes was induced, in Sprague-Dawley rats by streptozotocine (STZ) administration, 60 mg, i.v. Rats were divided into four groups; I: control, II: diabetics, III: control + SLD, IV: diabetics treated with SLD (15 mg/day). After three months, phenylephrine precontracted aortic rings were used to evaluate acetylcholine (ACh) and sodium nitroprusside (NPS) relaxation capacities. Light microscopy of aorta was done with several staining procedures. In vitro, SLD did not change smooth muscle tone in resting or phenylephrine precontracted aortic rings. In diabetic rats, ACh relaxation was 28.8-35.1% lower than in control rats. Diabetic rats treated with SLD showed aortic ACh relaxation similar to control rats. No significative statistical difference was found in endothelium-independent NPS relaxation, between the different groups. Light microscopy histological studies revealed important morphological alterations, particularly in intima and adventitia layers of aortic artery; those changes were dramatically reversed in SLD treated rats. Our experiments support the conclusion that SLD is a potential drug for improving endothelial dysfunction in diabetes.

[Estrogen therapy in menopause. Clinical study in evolution (KEEPS) to explain the risk-benefit dispute to administration]. / Terapia con estrógenos en la menopausia. Estudio clínico en evolución (KEEPS) para explicar la controversia del riesgo-beneficio de su administración.

The Kronos Early Estrogen Prevention Study (KEEPS) is a randomized clinical and controlled study, with the objective of clarifying the controversy that arisen previous studies about the risk-benefit factor with use of estrogens in postmenopausal women. Healthy women aged 42-58 years who are within 36 months of their last menstrual period have been recruited to receive either oral estrogens or patches of estradiol; in addition both groups are given oral micronized progesterone for 11 days of each month. Outcomes will be carotid intimae medial thickness and the accrual of coronary calcium; collaterally subrogate outcomes will be lipoproteins concentrations, coagulation markers, bone densitometry, mammography, skin characteristics and cognitive evaluation. The hypothesis consists in the presence of a window of therapeutic opportunity for the use of estrogens in low dose in healthy women with recent menopause.

Retrovirally mediated overexpression of glycosaminoglycan-deficient biglycan in arterial smooth muscle cells induces tropoelastin synthesis and elastic fiber formation in vitro and in neointimae after vascular injury.

Galactosamine-containing glycosaminoglycans (GAGs), such as the chondroitin sulfate chains of the proteoglycan versican, have been shown to inhibit elastogenesis. Another proteoglycan that may influence elastogenesis is biglycan, which possesses two GAG chains. To assess the importance of these chains on elastogenesis in blood vessels, rat aortic smooth muscle cells were transduced with a GAG-deficient biglycan cDNA-containing retroviral vector (LmBSN). Control cells were transduced with either biglycan or empty vector. Transduced cells were characterized in vitro and then seeded into balloon-injured rat carotid arteries to determine the effects on neointimal structure. Cultured cells overexpressing LmBSN showed marked up-regulation of tropoelastin and fibulin-5 mRNAs, increased amounts of desmosine and insoluble elastin, and increased deposition of elastic fibers as compared with empty vector- and biglycan-transduced cells. Conversely, collagen alpha(1) synthesis and the deposition of collagen fibers were both markedly decreased in LmBSN cultures. In vivo, neointimae formed from cells that overexpressed LmBSN and showed increased deposits of elastin that aggregated into parallel nascent fibers, generally arranged circumferentially. Neointimae that formed from cells with biglycan or empty vector contained fewer and less aggregated deposits of elastin. These findings suggest that the GAG chains of biglycan serve as inhibitors of elastin synthesis and assembly, and that biglycan can act as an important modulator of the composition of the extracellular matrix of blood vessels.