Kalkitoxin attenuates calcification of vascular smooth muscle cells via RUNX-2 signaling pathways.

BACKGROUND: Kalkitoxin (KT) is an active lipopeptide isolated from the cyanobacterium Lyngbya majuscula found in the bed of the coral reef. Although KT suppresses cell division and inflammation, KT's mechanism of action in vascular smooth muscle cells (VSMCs) is unidentified. Therefore, our main aim was to investigate the impact of KT on vascular calcification for the treatment of cardiovascular disease. OBJECTIVES: Using diverse calcification media, we studied the effect of KT on VSMC calcification and the underlying mechanism of this effect. METHODS: VSMC was isolated from the 6 weeks ICR mice. Then VSMCs were treated with different concentrations of KT to check the cell viability. Alizarin red and von Kossa staining were carried out to examine the calcium deposition on VSMC. Thoracic aorta of 6 weeks mice were taken and treated with different concentrations of KT, and H and E staining was performed. Real-time polymerase chain reaction and western blot were performed to examine KT's effect on VSMC mineralization. Calcium deposition on VSMC was examined with a calcium deposition quantification kit. RESULTS: Calcium deposition, Alizarin red, and von Kossa staining revealed that KT reduced inorganic phosphate-induced calcification phenotypes. KT also reduced Ca++-induced calcification by inhibiting genes that regulate osteoblast differentiation, such as runt-related transcription factor 2 (RUNX-2), SMAD family member 4, osterix, collagen 1α, and osteopontin. Also, KT repressed Ca2+-induced bone morphogenetic protein 2, RUNX-2, collagen 1α, osteoprotegerin, and smooth muscle actin protein expression. Likewise, Alizarin red and von Kossa staining showed that KT markedly decreased the calcification of ex vivo ring formation in the mouse thoracic aorta. CONCLUSIONS: This experiment demonstrated that KT decreases vascular calcification and may be developed as a new therapeutic treatment for vascular calcification and arteriosclerosis.

A feline-focused review of chronic kidney disease-mineral and bone disorders - Part 2: Pathophysiology of calcium disorder and extraosseous calcification.

Derangements in mineral metabolism are one of the main entities in chronic kidney disease-mineral and bone disorder (CKD-MBD). This is the second of a two-part review of the physiology and pathophysiology of calcium homeostasis in feline CKD-MBD. While dysregulation in calcium homeostasis is known to contribute to the development of vascular calcification in CKD, evidence characterising the relationship between serum calcium concentration and nephrocalcinosis and nephrolithiasis is limited. Recently, fibroblast growth factor 23 (FGF23) and α-Klotho have gained increased research interest and been shown to be important biomarkers for the prediction of CKD progression in human patients. However, conflicting evidence exists on their role in calcium homeostasis and vascular and soft tissue calcification. This review details the pathophysiology of calcium disorders associated with CKD-MBD and its implications on vascular and soft tissue mineralisation in human and feline patients. Further prospective studies investigating the clinical consequences of calcium disturbances in cats with CKD are warranted and this may provide additional insight into the pathophysiology of feline CKD-MBD.

Vascular mineralization in the brain of horses.

Vascular mineralization (siderocalcinosis) in the brain of horses has been usually assumed to be an incidental age-related finding with no clinic significance. In the present study, eight 15-32-year-old horses of different breeds with cerebral siderocalcinosis were studied. Four of these horses had acute and severe central nervous system clinical signs of unknown etiology, 2 horses had neurological signs of known cause, and 2 horses did not have neurological signs. Gross examination of the brains in 4 animals revealed symmetrical foci of malacia in the cerebellar white matter. Histologically, moderate to severe mineralization of blood vessels and parenchyma were observed in all 8 horses, occasionally associated with necrosis of the adjacent tissue. Some horses were tested by virus isolation, polymerase chain reaction, immunohistochemistry, and serology to investigate Rabies virus; West Nile virus; Equid herpesvirus 1 and 4; Eastern, Western, Venezuelan, and Saint Louis encephalitis virus; and Sarcocystis neurona infection. These tests were negative in all samples analyzed. Brain cholinesterase activity and heavy metal screening were also unremarkable. The significance of the vascular and parenchymal mineralization in the brains of some of these horses remains undetermined. However, the severity of the lesions observed in the brains of some of the animals in the present study, coupled with the negative results for other common causes of neurological disease in horses, suggests a possible relationship between siderocalcinosis and the clinical signs observed.

Efecto del estrés oxidativo sobre la calcificación vascular a través del microARN-377 / The effect of oxidative stress on vascular calcification through microRNA-377

Introducción: El estrés oxidativo ha sido implicado en el desarrollo y la progresión de la calcificación vascular (CV); sin embargo, aún existen interrogantes sobre esta asociación causal. Objetivo: Analizar en un modelo experimental de insuficiencia renal crónica (IRC) el efecto del estrés oxidativo sobre el desarrollo y la progresión de la CV, evaluando la implicación del microARN-377 (miR-377). Material y métodos: Se estudiaron 2 grupos de ratas Wistar con IRC. El grupo 1 recibió dieta normal en fósforo (IRC+PN). El grupo 2 recibió dieta con alto fósforo (IRC+PA). Se incluyó un grupo de ratas Sham. Trascurridas 20 semanas, las ratas fueron sacrificadas. Resultados: El fósforo y la parathormona séricos no aumentaron en el grupo IRC+PA respecto al IRC+PN, pero sí los niveles de factor de crecimiento fibrobástico 23 (FGF23). En el grupo IRC+PN aumentó tres veces el contenido aórtico de calcio respecto al grupo Sham, un aumento 17 veces superior en el grupo IRC+PA, donde la densidad mineral ósea en tibia proximal descendió significativamente. En el grupo IRC+PN la expresión del miR-377 disminuyó un 65%, sin efecto adicional de la dieta con alto contenido en fósforo. En el grupo IRC+PN aumentó 3 veces la expresión proteica de superóxido dismutasa 2 mitocondrial (SOD-2), y en el grupo IRC+PA lo hizo hasta 6 veces. Conclusiones: La IRC con o sin alto contenido en fósforo en la dieta desencadenó el descenso del miR-377. El exceso de fósforo incrementó la SOD-2 como mecanismo compensador para frenar el estrés oxidativo y el daño vascular. Controlar el contenido en fósforo en la dieta cuando la función renal se ve comprometida permitirá aminorar el daño vascular producido como consecuencia, entre otros factores, del estrés oxidativo (AU)

Introduction: Oxidative stress has been implicated in the development and progression of vascular calcification (VC). However, this causal association remains a matter of controversy. Objective: To analyze in an experimental model of chronic renal failure (CRF), the effect of oxidative stress on the development and progression of the VC, assessing the implication of microRNA-377 (miR-377). Material and methods: Two groups of Wistar rats with CRF were studied. Group 1 received normal diet in phosphorus (CRF+NP). Group 2 received a high phosphorus (CRF+HP) diet. A group of sham rats was included. After 20 weeks, the rats were sacrificed. Results: Serum phosphorus and parathormone did not increase in the CRF+HP group compared to CRF+NP, but fibroblast growth factor 23 (FGF23) levels significantly increased. In the CRF+NP group, aortic calcium content increased three-fold over the sham group, a 17-fold increase in the CRF+HP group, where the bone mineral density in the proximal tibia decreased significantly. In the IRC+NP group, the expression of miR-377 decreased by 65%, with no additional effect detected of the diet with high phosphorus content. In the IRC+NP group, the protein expression of mitochondrial superoxide dismutase 2 (SOD-2) increased 3-fold, and in the IRC+HP group it increased up to 6-fold. Conclusions: CRF, with or without high phosphorus dietary content, triggered the descent of miR-377. Excess phosphorus increased SOD-2 as a compensatory mechanism to curb oxidative stress and vascular damage. Controlling phosphorus content in the diet when the renal impairment function is compromised will reduce the vascular damage produced due oxidative stress, among other factors (AU)

Nanopartículas calcificantes como factor etiológico del desarrollo de hiperplasia y calcificación vascular / Calcifying nanoparticles in the aetiology of hyperplasia and calcification

OBJETIVO: Nanopartículas calcificantes (NP) se han detectado recientemente en muestras arteriales humanas y parecen estar involucradas en el proceso de calcificación. Este estudio fue diseñado para probar la hipótesis de que las NP de origen humano podrían agravar la respuesta a la lesión arterial endotelial e inducir la calcificación vascular. MÉTODOS: La arteria carótida derecha de 24 conejos neozelandeses fue lesionada con un balón de angioplastia. Los animales fueron perfundidos por vía intravenosa con solución salina (100 ml) durante el experimento y se dividieron en 3 grupos: grupo A, control; grupo B, expuesto a NP (2 ml) obtenidas a partir de válvulas aórticas calcificadas y el grupo C, expuesto a NP (2 ml) y tratado después de la operación con atorvastatina (2,5 mg/kg/24 h). A los 30 días, los animales fueron sacrificados y se extirparon las 2 arterias carótidas, que fueron examinadas histológicamente. Análisis bioquímicos de sangre fueron realizados durante el estudio. RESULTADOS: El área de hiperplasia intimal fue significativamente mayor en la arteria carótida derecha lesionada en comparación con la arteria carótida izquierda no operada, en todos los grupos. No hubo variación significativa en la zona medial entre los animales. Morfométricamente, la relación de íntima/media (IMR) fue significativamente mayor en las carótidas dañadas en comparación con los controles. Un aumento significativo de IMR se encontró en el grupo B (1,81 ± 0,41) en comparación con el grupo A (0,38 ± 0,59; p = 0,004) o el grupo C (0,89 ± 0,79; p = 0,035). Las diferencias entre los grupos C y A no fueron significativas (p = 0,064). Se observaron calcificaciones en 6 animales, todos los cuales habían sido expuestos a NP (4 en el grupo B, 2 en el grupo C, p = 0,027). Los niveles plasmáticos de colesterol y triglicéridos se mantuvieron estables. CONCLUSIONES: Este estudio confirma la capacidad de las NP de origen humano de acelerar la hiperplasia y estimular la calcificación de zonas arteriales endoteliales previamente dañadas. Su administración sistémica resultó inofensiva en las arterias sanas. La atorvastatina demostró la capacidad de ralentizar este proceso

OBJECTIVE: Calcifying nanoparticles (NP) have been detected recently in calcified human arterial specimens, and are involved in the process of calcification. This study was designed to test the hypothesis that human-derived NP could worsen the response to arterial endothelial injury and induce vascular calcification. METHODS: The right carotid artery of 24 New Zealand rabbits was injured with an angioplasty balloon. Animals were perfused intravenously with saline (100 mL) during the experiment and divided into 3 groups: group A, control; group B, exposed to NP (2 mL) obtained from calcified aortic valves; and group C, exposed to NP (2 mL) and treated post-operatively with atorvastatin (2.5 mg/kg/24 h). At 30 days, both carotid arteries were removed and examined histologically. Blood measurements were monitored during the study. RESULTS: The intimal hyperplasia area was significantly larger in the injured right carotid artery compared with the left un-operated carotid artery in all groups. There was no significant variation in medial area between groups. Morphometrically, the intima/media ratio (IMR) was significantly higher in damaged carotids compared with controls. A significant increase in the IMR was found in group B (1.81 ± 0.41) compared with group A (0.38 ± 0.59; P=.004) or group C (0.89 ± 0.79; P=.035). Differences between groups C and A were not significant (P=.064). Calcifications were observed in 6 animals, all of which had been exposed to NP (4 in group B, and 2 in group C, P=.027). Plasma levels of cholesterol and triglycerides remained stable. CONCLUSIONS: his study confirms the ability of systemic inoculation of human-derived NP to accelerate hyperplasia and stimulate calcification in localised areas of arteries previously submitted to endothelial damage, while it was harmless in healthy arteries. Atorvastatin was demonstrated to slow down this process