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Sex hormones play a pivotal role as endocrine hormones that exert profound effects on the biological characteristics and vascular function of vascular smooth muscle cells (VSMCs). By modulating intracellular signaling pathways, activating nuclear receptors, and regulating gene expression, sex hormones intricately influence the morphology, function, and physiological state of VSMCs, thereby impacting the biological properties of vascular contraction, relaxation, and growth. Increasing evidence suggests that abnormal phenotypic changes in VSMCs contribute to the initiation of vascular diseases, including atherosclerosis. Therefore, understanding the factors governing phenotypic alterations in VSMCs and elucidating the underlying mechanisms can provide crucial insights for refining interventions targeted at vascular diseases. Additionally, the varying levels of different types of sex hormones in the human body, influenced by sex and age, may also affect the phenotypic conversion of VSMCs. This review aims to explore the influence of sex hormones on the phenotypic switching of VSMCs and the development of associated vascular diseases in the human body.

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Redox processes can modulate vascular pathophysiology. The endoplasmic reticulum redox chaperone protein disulfide isomerase A1 (PDIA1) is overexpressed during vascular proliferative diseases, regulating thrombus formation, endoplasmic reticulum stress adaptation, and structural remodeling. However, both protective and deleterious vascular effects have been reported for PDIA1, depending on the cell type and underlying vascular condition. Further understanding of this question is hampered by the poorly studied mechanisms underlying PDIA1 expression regulation. Here, we showed that PDIA1 mRNA and protein levels were upregulated (average 5-fold) in the intima and media/adventitia following partial carotid ligation (PCL). Our search identified that miR-204-5p and miR-211-5p (miR-204/211), two broadly conserved miRNAs, share PDIA1 as a potential target. MiR-204/211 was downregulated in vascular layers following PCL. In isolated endothelial cells, gain-of-function experiments of miR-204 with miR mimic decreased PDIA1 mRNA while having negligible effects on markers of endothelial activation/stress response. Similar effects were observed in vascular smooth muscle cells (VSMCs). Furthermore, PDIA1 downregulation by miR-204 decreased levels of the VSMC contractile differentiation markers. In addition, PDIA1 overexpression prevented VSMC dedifferentiation by miR-204. Collectively, we report a new mechanism for PDIA1 regulation through miR-204 and identify its relevance in a model of vascular disease playing a role in VSMC differentiation. This mechanism may be regulated in distinct stages of atherosclerosis and provide a potential therapeutic target.

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Calcium signaling in vascular endothelial cells (ECs) and smooth muscle cells (VSMCs) is essential for the regulation of vascular tone. However, the changes to intracellular Ca2+ concentrations are often influenced by sex differences. Furthermore, a large body of evidence shows that sex hormone imbalance leads to dysregulation of Ca2+ signaling and this is a key factor in the pathogenesis of cardiovascular diseases. In this review, the effects of estrogens and androgens on vascular calcium-handling proteins are discussed, with emphasis on the associated genomic or nongenomic molecular mechanisms. The experimental models from which data were collected were also considered. The review highlights 1) in female ECs, transient receptor potential vanilloid 4 (TRPV4) and mitochondrial Ca2+ uniporter (MCU) enhance Ca2+-dependent nitric oxide (NO) generation. In males, only transient receptor potential canonical 3 (TRPC3) plays a fundamental role in this effect. 2) Female VSMCs have lower cytosolic Ca2+ levels than males due to differences in the activity and expression of stromal interaction molecule 1 (STIM1), calcium release-activated calcium modulator 1 (Orai1), calcium voltage-gated channel subunit-α1C (CaV1.2), Na+-K+-2Cl- symporter (NKCC1), and the Na+/K+-ATPase. 3) When compared with androgens, the influence of estrogens on Ca2+ homeostasis, vascular tone, and incidence of vascular disease is better documented. 4) Many studies use supraphysiological concentrations of sex hormones, which may limit the physiological relevance of outcomes. 5) Sex-dependent differences in Ca2+ signaling mean both sexes ought to be included in experimental design.

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INTRODUCTION: Thoracic aortic aneurysm is a potentially fatal disease with a strong genetic contribution. The dysfunction of vascular smooth muscle cells (VSMCs) contributes to the formation of this aneurysm. Although previous studies suggested that long non-coding ribonucleic acid (RNA) hypoxia inducible factor 1 α-antisense RNA 1 (HIF1A-AS1) exerted a vital role in the progression and pathogenesis of thoracic aortic aneurysm, we managed to find a new regulatory mechanism of HIF1A-AS1 in VSMCs via transcriptomics. METHODS: Cell viability was detected by the cell counting kit-8 assay. Cell apoptosis was assessed by Annexin V-fluorescein isothiocyanate/propidium iodide double staining. Transwell migration assay and wound healing assay were performed to check the migration ability of HIF1A-AS1 on VSMCs. The NextSeq XTen system (Illumina) was used to collect RNA sequencing data. Lastly, reverse transcription-quantitative polymerase chain reaction confirmed the veracity and reliability of RNA-sequencing results. RESULTS: We observed that overexpressing HIF1A-AS1 successfully promoted apoptosis, significantly altered cell cycle distribution, and greatly attenuated migration in VSMCs, further highlighting the robust promoting effects of HIF1A-AS1 to thoracic aortic aneurysm. Moreover, transcriptomics was implemented to uncover its underlying mechanism. A total of 175 differently expressed genes were identified, with some of them enriched in apoptosis, migration, and cell cycle-related pathways. Intriguingly, some differently expressed genes were noted in vascular development or coagulation function pathways. CONCLUSION: We suggest that HIF1A-AS1 mediated the progression of thoracic aortic aneurysm by not only regulating the function of VSMCs, but also altering vascular development or coagulation function.

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AIMS: Our objective was to study the vascular smooth muscle cells (VSMC) osteoblastic transdifferentiation in AGE exposed cells or those from diabetic animals, and its response to metformin treatment. METHODS: VSMC were obtained from non-diabetic rats, grown with or without AGE; while VSMC of in vivo-ex vivo studies were obtained from non-diabetic control animals (C), diabetic (D), C treated with metformin (M) and D treated with metformin (D-M). We studied the osteoblastic differentiation by evaluating alkaline phosphatase (ALP), type I collagen (Col) and mineral deposit. RESULTS: In vitro, AGE increased proliferation, migration, and osteoblastic differentiation of VSMC. Metformin cotreatment prevented the AGE induced proliferation and migration. Both AGE and metformin stimulated the expression of ALP and Col. AGE induced mineralization was prevented by metformin. VSMC from D expressed a higher production of Col and ALP. Those from D-M showed an ALP increase vs C and M, and a partial decrease vs D. Cultured in osteogenic medium, ALP, Col and mineralization increased in D vs C, remained unchanged in M, and were prevented in D-M animals. CONCLUSION: Both AGE and DM favor VSMC differentiation towards the osteogenic phenotype and this effect can be prevented by metformin.

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In post-menopausal women, aged individuals, and patients with diabetes mellitus or chronic renal disease, bone mineral density (BMD) decreases while the vasculature accumulates arterial calcifications (ACs). AC can be found in the tunica intima and/or in the tunica media. Prospective studies have shown that patients with initially low BMD and/or the presence of fragility fractures have at follow-up a significantly increased risk for coronary and cerebrovascular events and for overall cardiovascular mortality. Similarly, patients presenting with abdominal aorta calcifications (an easily quantifiable marker of vascular pathology) show a significant decrease in the BMD (and an increase in the fragility) of bones irrigated by branches of the abdominal aorta, such as the hip and lumbar spine. AC induction is an ectopic tissue biomineralization process promoted by osteogenic transdifferentiation of vascular smooth muscle cells as well as by local and systemic secreted factors. In many cases, the same regulatory molecules modulate bone metabolism but in reverse. Investigation of animal and in vitro models has identified several potential mechanisms for this reciprocal bone-vascular regulation, such as vitamin K and D sufficiency, advanced glycation end-products-RAGE interaction, osteoprotegerin/RANKL/RANK, Fetuin A, oestrogen deficiency and phytooestrogen supplementation, microbiota and its relation to diet, among others. Complete elucidation of these potential mechanisms, as well as their clinical validation via controlled studies, will provide a basis for pharmacological intervention that could simultaneously promote bone and vascular health.

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INTRODUCTION: Increased matrix metalloproteinase (MMP)-2 activity contributes to increase vascular smooth muscle cell (VSMC) proliferation in the aorta in early hypertension by cleaving many proteins of the extracellular matrix. Cleaved products from type I collagen may activate focal adhesion kinases (FAK) that trigger migration and proliferation signals in VSMC. We therefore hypothesized that increased activity of MMP-2 proteolyzes type I collagen in aortas of hypertensive rats, and thereby, induces FAK activation, thus leading to increased VSMC proliferation and hypertrophic remodeling in early hypertension. METHODS: Male Sprague-Dawley rats were submitted to renovascular hypertension by the two kidney-one clip (2K1C) model and treated with doxycycline (30 mg/kg/day) by gavage from the third to seventh-day post-surgery. Controls were submitted to sham surgery. Systolic blood pressure (SBP) was measured daily by tail-cuff plethysmography and the aortas were processed for zymography and Western blot for MMP-2, pFAK/FAK, integrins and type I collagen. Mass spectrometry, morphological analysis and Ki67 immunofluorescence were also done to identify collagen changes and VSMC proliferation. A7r5 cells were stimulated with collagen and treated with the MMP inhibitors (doxycycline or ARP-100), and with the FAK inhibitor PND1186 for 24 h. Cells were lysed and evaluated by Western blot for pFAK/FAK. RESULTS: 2K1C rats developed elevated SBP in the first week as well as increased expression and activity of MMP-2 in the aorta (p < 0.05 vs. Sham). Treatment with doxycycline reduced both MMP activity and type I collagen proteolysis in aortas of 2K1C rats (p < 0.05). Increased pFAK/FAK and increased VSMC proliferation (p < 0.05 vs. Sham groups) were also seen in the aortas of 2K1C and doxycycline decreased both parameters (p < 0.05). Higher proliferation of VSMC contributed to hypertrophic remodeling as seen by increased media/lumen ratio and cross sectional area (p < 0.05 vs Sham groups). In cell culture, MMP-2 cleaves collagen, an effect reversed by MMP inhibitors (p < 0.05). Increased levels of pFAK/FAK were observed when collagen was added in the culture medium (p < 0.05 vs control) and MMP and FAK inhibitors reduced this effect. CONCLUSIONS: Increase in MMP-2 activity proteolyzes type I collagen in the aortas of 2K1C rats and contributes to activate FAK and induces VSMC proliferation during the initial phase of hypertension.

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Type 2 Diabetes Mellitus (T2DM) is a rapidly rising disease with cardiovascular complications constituting the most common cause of death among diabetic patients. Chronic hyperglycemia can induce vascular dysfunction through damage of the components of the vascular wall, such as vascular smooth muscle cells (VSMCs), which regulate vascular tone and contribute to vascular repair and remodeling. These functions are dependent on intracellular Ca2+ changes. The mechanisms by which T2DM affects Ca2+ handling in VSMCs still remain poorly understood. Therefore, the objective of this study was to determine whether and how T2DM affects Ca2+ homeostasis in VSMCs. We evaluated intracellular Ca2+ signaling in VSMCs from Zucker Diabetic Fatty rats using Ca2+ imaging with Fura-2/AM. Our results indicate that T2DM decreases Ca2+ release from the sarcoplasmic reticulum (SR) and increases the activity of store-operated channels (SOCs). Moreover, we were able to identify an enhancement of the activity of the main Ca2+ extrusion mechanisms (SERCA, PMCA and NCX) during the early stage of the decay of the ATP-induced Ca2+ transient. In addition, we found an increase in Ca2+ entry through the reverse mode of NCX and a decrease in SERCA and PMCA activity during the late stage of the signal decay. These effects were appreciated as a shortening of ATP-induced Ca2+ transient during the early stage of the decay, as well as an increase in the amplitude of the following plateau. Enhanced cytosolic Ca2+ activity in VSMCs could contribute to vascular dysfunction associated with T2DM.

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ABSTRACT Introduction: Thoracic aortic aneurysm is a potentially fatal disease with a strong genetic contribution. The dysfunction of vascular smooth muscle cells (VSMCs) contributes to the formation of this aneurysm. Although previous studies suggested that long non-coding ribonucleic acid (RNA) hypoxia inducible factor 1 α-antisense RNA 1 (HIF1A-AS1) exerted a vital role in the progression and pathogenesis of thoracic aortic aneurysm, we managed to find a new regulatory mechanism of HIF1A-AS1 in VSMCs via transcriptomics. Methods: Cell viability was detected by the cell counting kit-8 assay. Cell apoptosis was assessed by Annexin V-fluorescein isothiocyanate/propidium iodide double staining. Transwell migration assay and wound healing assay were performed to check the migration ability of HIF1A-AS1 on VSMCs. The NextSeq XTen system (Illumina) was used to collect RNA sequencing data. Lastly, reverse transcription-quantitative polymerase chain reaction confirmed the veracity and reliability of RNA-sequencing results. Results: We observed that overexpressing HIF1A-AS1 successfully promoted apoptosis, significantly altered cell cycle distribution, and greatly attenuated migration in VSMCs, further highlighting the robust promoting effects of HIF1A-AS1 to thoracic aortic aneurysm. Moreover, transcriptomics was implemented to uncover its underlying mechanism. A total of 175 differently expressed genes were identified, with some of them enriched in apoptosis, migration, and cell cycle-related pathways. Intriguingly, some differently expressed genes were noted in vascular development or coagulation function pathways. Conclusion: We suggest that HIF1A-AS1 mediated the progression of thoracic aortic aneurysm by not only regulating the function of VSMCs, but also altering vascular development or coagulation function.

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Abstract Background: To evaluate the effect of T-helper 17 (Th17) cells and Th9 cells on the activation of dermal vascular smooth muscle cells (DVSMCs) in systemic scleroderma (SSc) and regulation of tanshinone IIA. Methods: The expression of interleukin 17 receptor (IL-17R) and interleukin 9 receptor (IL-9R) in the skin of SSc patients was assessed by immunofluorescence. The expression of IL-9 and IL-9R mRNA in peripheral blood mononuclear cells (PBMCs) of SSc patients were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The proportion of Th9 cells in PBMCs of SSc patients was sorted by flow cytometry. The effect of IL-9 on the differentiation of Th17 and IL-17 on that of Th9 was detected by flow cytometry. The proportion of Th9 and Th17 cells in SSc patients was detected by flow cytometry. The level of collagen I, III, α-SMA, IL-9R, IL-17R, JNK, P38, and ERK were analyzed using western blot (WB). Results: Th9 cells were highly expressed in SSc. IL-9 stimulated the differentiation of immature T cells into Th17 cells. IL-17 induced the differentiation of immature T cells intoTh9 cells. Tanshinone IIA inhibited the differentiation of immature T lymphocytes into Th17 and Th9. WB showed that the combined action of IL-17 and IL-9 upregulated the inflammation and proliferation of DVSMCs. Anti-IL17, anti-IL9, and tanshinone IIA inhibited the functional activation of DVSMCs. Study limitations: For Th17, Th9 and vascular smooth muscle cells, the study on the signal pathway of their interaction is not thorough enough. More detailed studies are needed to explore the mechanism of cell-cell interaction. Conclusions: The current results suggested that Th17 and Th9 cells induced the activation of DVSMCs in SSc through crosstalk in vitro, and tanshinone IIA inhibited the process.

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BACKGROUND: To evaluate the effect of T-helper 17 (Th17) cells and Th9 cells on the activation of dermal vascular smooth muscle cells (DVSMCs) in systemic scleroderma (SSc) and regulation of tanshinone IIA. METHODS: The expression of interleukin 17 receptor (IL-17R) and interleukin 9 receptor (IL-9R) in the skin of SSc patients was assessed by immunofluorescence. The expression of IL-9 and IL-9R mRNA in peripheral blood mononuclear cells (PBMCs) of SSc patients were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The proportion of Th9 cells in PBMCs of SSc patients was sorted by flow cytometry. The effect of IL-9 on the differentiation of Th17 and IL-17 on that of Th9 was detected by flow cytometry. The proportion of Th9 and Th17 cells in SSc patients was detected by flow cytometry. The level of collagen I, III, α-SMA, IL-9R, IL-17R, JNK, P38, and ERK were analyzed using western blot (WB). RESULTS: Th9 cells were highly expressed in SSc. IL-9 stimulated the differentiation of immature T cells into Th17 cells. IL-17 induced the differentiation of immature T cells into Th9 cells. Tanshinone IIA inhibited the differentiation of immature T lymphocytes into Th17 and Th9. WB showed that the combined action of IL-17 and IL-9 upregulated the inflammation and proliferation of DVSMCs. Anti-IL17, anti-IL9, and tanshinone IIA inhibited the functional activation of DVSMCs. STUDY LIMITATIONS: For Th17, Th9 and vascular smooth muscle cells, the study on the signal pathway of their interaction is not thorough enough. More detailed studies are needed to explore the mechanism of cell-cell interaction. CONCLUSIONS: The current results suggested that Th17 and Th9 cells induced the activation of DVSMCs in SSc through crosstalk in vitro, and tanshinone IIA inhibited the process.

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RESUMEN Introducción: Una de las causas propuestas del síndrome INOCA (por sus siglas en inglés: Ischemia with Non-Obstructive Coronary Arteries) es la disfunción microvascular (DMV), la cual puede evaluarse en forma no invasiva, mediante la cuantificación del flujo sanguíneo miocárdico (FSM) y la reserva de flujo miocárdica (RFM). Las imágenes de perfusión miocárdica (IPM) y dinámicas con CZT-SPECT en reposo - dipiridamol - y prueba de frio (PF), permiten establecer la presencia de DMV evaluando diferentes mecanismos fisiopatológicos: endotelio independiente o dependiente, respectivamente. Objetivos: Evaluar la utilidad de CZT-SPECT en el diagnóstico de DMV y los diferentes mecanismos patológicos involucrados, en pacientes con diagnóstico de INOCA. Material y métodos: Se incluyeron en forma prospectiva 93 pacientes consecutivos con diagnóstico de INOCA, a los que se les realizó IPM e imágenes dinámicas con CZT-SPECT en reposo-dipiridamol-PF. El FSM se cuantificó con el software 4DM. Se consideró respuesta anormal al dipiridamol una RFM menor a 2 y a la variación del FSM (∆FSM) menor a 1,5 con PF. Se definió DMV a la presencia de una o ambas respuestas anormales. Resultados: El CZT-SPECT detectó DMV en un 85% (n=79) de los pacientes con INOCA. El 42% tuvo respuesta anormal con ambos apremios mientras que el 43% restante, mostró una respuesta alterada del FSM sólo con PF. Conclusiones: El uso de CZT-SPECT empleando ambos apremios, permitió evaluar diferentes mecanismos fisiopatológicos que causan DMV presente en la mayoría de los pacientes con INOCA.

ABSTRACT Background: One of the causes of INOCA (Ischemia with Non- Obstructive Coronary Arteries) is microvascular dysfunction (MVD), which can be noninvasively assessed through the quantification of myocardial blood flow (MBF) and myocardial flow reserve (MFR). Dynamic myocardial perfusion imaging (MPI) by CZT-SPECT at rest, with dipyridamole stress test and cold pressor test (CPT) can establish the presence of two different pathophysiological mechanisms of MVD: endothelium-independent or endothelium-dependent, respectively. Objectives: The aim of this study was to evaluate the usefulness of CZT-SPECT for the diagnosis of MVD and the different mechanisms involved in patients with INOCA. Materials and Methods : A total of 93 consecutive INOCA patients were prospectively included and underwent dynamic MPI with CZT-SPECT at rest and with dipyridamole stress test and CPT. THe MBF was quantified using 4DM® software. A MFR response to dipyridamole <2, and changes in MBF (∆MBF) <1.5 with CPT were considered abnormal responses. MVD was defined in the presence of one abnormal response or both. Results: CZT-SPECT detected MVD in 85% (n=79) of the patients with INOCA. Forty-two percent had an abnormal response to both stressors while 43% presented an abnormal response of MBF only with CPT. Conclusion: The use of CZT-SPECT with both stress tests allowed the evaluation of different possible pathophysiological mechanisms of MVD present in most patients with INOCA.

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Vascular mineralization is a hallmark of enzootic calcinosis. Histopathological, ultrastructural, and immunohistochemical investigations were performed on the external carotid arteries of seven sheep naturally poisoned by Nierembergia veitchii. Histologically, moderate to marked hyperplasia of the tunica intima was observed without mineralization. The tunica media exhibited mild to severe mineralization and osteochondroid metaplasia. Sheep with enzootic calcinosis showed arterial overexpression of osteopontin and tissue-nonspecific alkaline phosphatase and immunolabeling for osteonectin and osteocalcin in both intima and media layers of the tested arteries. The main ultrastructural finding in the tunica media was a marked phenotypic change of vascular smooth muscle cells from a contractile phenotype (VSMC-C) into a synthetic phenotype (VSMC-S). In the tunica media, VSMC-S produced matrix and extracellular vesicles, forming mineralizable granules associated with arterial mineralization. VSMC-S were also present in the tunica intima, but matrix and extracellular vesicles and mineralization were not observed. The absence of matrix and extracellular vesicles in the intimal hyperplasia, even in the presence of noncollagenous bone proteins, tissue-nonspecific alkaline phosphatase, and vitamin D receptors, reinforces the hypothesis that the presence of matrix and extracellular vesicles are crucial for the development of vascular mineralization in enzootic calcinosis. It is proposed that the two different VSMC-S phenotypes in calcinosis are due to the expression of at least two genetically different types of these cells induced by the action of 1,25(OH)2D3.

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In mesenteric arteries (MAs), aldosterone (ALDO) binds to the endogenous mineralocorticoid receptor (MR) and increases the expression of the voltage-gated L-type Cav1.2 channel, an essential ion channel for vascular contraction, sarcoplasmic reticulum (SR) Ca2+ store refilling, and Ca2+ spark generation. In mesenteric artery smooth muscle cells (MASMCs), Ca2+ influx through Cav1.2 is the indirect mechanism for triggering Ca2+ sparks. This process is facilitated by plasma membrane-sarcoplasmic reticulum (PM-SR) nanojunctions that drive Ca2+ from the extracellular space into the SR via Sarco/Endoplasmic Reticulum Ca2+ (SERCA) pump. Ca2+ sparks produced by clusters of Ryanodine receptors (RyRs) at PM-SR nanodomains, decrease contractility by activating large-conductance Ca2+-activated K+ channels (BKCa channels), which generate spontaneous transient outward currents (STOCs). Altogether, Cav1.2, SERCA pump, RyRs, and BKCa channels work as a functional unit at the PM-SR nanodomain, regulating intracellular Ca2+ and vascular function. However, the effect of the ALDO/MR signaling pathway on this functional unit has not been completely explored. Our results show that short-term exposure to ALDO (10 nM, 24 h) increased the expression of Cav1.2 in rat MAs. The depolarization-induced Ca2+ entry increased SR Ca2+ load, and the frequencies of both Ca2+ sparks and STOCs, while [Ca2+]cyt and vasoconstriction remained unaltered in Aldo-treated MAs. ALDO treatment significantly increased the mRNA and protein expression levels of the SERCA pump, which counterbalanced the augmented Cav1.2-mediated Ca2+ influx at the PM-SR nanodomain, increasing SR Ca2+ content, Ca2+ spark and STOC frequencies, and opposing to hyperpolarization-induced vasoconstriction while enhancing Acetylcholine-mediated vasorelaxation. This work provides novel evidence for short-term ALDO-induced upregulation of the functional unit comprising Cav1.2, SERCA2 pump, RyRs, and BKCa channels; in which the SERCA pump buffers ALDO-induced upregulation of Ca2+ entry at the superficial SR-PM nanodomain of MASMCs, preventing ALDO-triggered depolarization-induced vasoconstriction and enhancing vasodilation. Pathological conditions that lead to SERCA pump downregulation, for instance, chronic exposure to ALDO, might favor the development of ALDO/MR-mediated augmented vasoconstriction of mesenteric arteries.

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BACKGROUND: Acetaminophen (APAP) is the most widely used analgesic and antipyretic in the world. However, in high or continuous doses, it can cause serious side effects including blood pressure variability and cardiovascular injuries, which are barely explored. This study aimed to evaluate the acute effect of APAP treatment on vascular tone focused on the blocking of Ca2+ channels. METHODS: Rats were treated with APAP orally by gavage (500 mg/kg/single dose). After 12 h, the aorta was isolated for vascular reactivity studies in an isolated organ bath. Vascular contraction and relaxation were measured after different stimuli. Moreover, molecular docking studies were performed to evaluate the action of NAPQI (APAP metabolite) on L-type calcium channels. RESULTS: Phenylephrine-induced maximal vascular contraction was reduced in the APAP group (138.4 ± 9.2%) compared to the control group (172.2 ± 11.1%). APAP treatment significantly reduced contraction induced by Ca2+ influx stimulated with phenylephrine or KCl and reduced contraction mediated by Ca2+ released from the sarcoplasmic reticulum induced by caffeine. There was no difference in vascular relaxation induced by acetylcholine or sodium nitroprusside. Computational molecular docking demonstrated that NAPQI is capable of blocking L-type Ca2+ channels (Cav1.2), which would limit the influx of Ca2+. CONCLUSION: These results suggest that APAP treatment causes an anticontractile effect in rat aorta, possibly by blocking the influx of Ca2+ through L-type channels (Cav1.2).

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RESUMEN Introducción: El Camphenol Plus es un derivado clorofenólico empleado como medicación intraconducto durante los tratamientos pulporradiculares en Estomatología. Son escasos los reportes científicos sobre el papel de los canales de iones potasio en la dinámica contráctil del músculo liso arterial inducida por dicho medicamento. Objetivo: Determinar el papel de los canales de iones potasio en la dinámica contráctil del músculo liso arterial inducida por Camphenol Plus. Método: Se realizó una investigación experimental preclínica en el Instituto de Fisiología "Oscar Langerdorff", Facultad de Medicina, Universidad de Rostock, Alemania, entre octubre y diciembre de 2018, con el empleo de 30 anillos de aorta obtenidos de 10 ratas Wistar (n=10). Las preparaciones biológicas se colocaron en baño de órganos y se preactivaron con solución Krebs concentrada en iones potasio, registrándose luego la tensión desarrollada por el músculo liso vascular tras la adición de soluciones de Camphenol Plus durante diferentes intervalos de tiempo. Se utilizaron las pruebas de Wilcoxon y U de Mann-Whitney. Resultados: El 31,4 % de la musculatura lisa vascular se relajó por acción del Camphenol Plus tras la preactivación con solución Krebs concentrada en iones potasio. El mayor descenso del tono vascular se produjo con el uso de soluciones del medicamento al 7 % entre el primer y tercer minutos. Conclusiones : El efecto vasorrelajante in vitro producido por Camphenol Plus sobre el músculo liso arterial está mediado por canales de iones potasio sensibles a voltaje, a calcio y a trifosfato de adenosina del endotelio vascular y el sarcolema.

ABSTRACT Introduction: Camphenol Plus is a chlorophenolic derivative commonly used as an intra - duct medication for pulporadicular treatments in Dentistry. Scientific reports about the use of this medication on the role of potassium ion channels in the contractile dynamics of induced arterial smooth muscle are low. Objective: To determine the role of potassium ion channels in the contractile dynamics of Camphenol Plus - induced arterial smooth muscle. Method: A preclinical experimental investigation was performed at the "Oscar Langerdorff" Institute of Physiology, Rostock University Medical Center, Rostock, Germany, between October and December 2018. A total of 30 aortic rings obtained from 10 Wistar rats (n=10) were used. The biological preparations were placed in an organ bath and preactivated with Krebs solution concentrated in potassium ions, afterwards it was recorded the tension developed by the vascular smooth muscle after applying the Camphenol Plus solutions in different time intervals. The Mann-Whitney U test and Wilcoxon test were applied. Results: The 31.4% of vascular smooth muscle was relaxed by the effect of Camphenol Plus after preactivation with Krebs solution concentrated in potassium ions. The greatest decrease in vascular tone occurred between the first and third minutes after the use of the drug solutions prepared at 7 %. Conclusions: The in vitro vasorelaxant effect produced by the Camphenol Plus medication on arterial smooth muscle is mediated by the potassium ion channels sensitive to voltage, calcium and the adenosine triphosphate of the vascular endothelium and sarcolemma.

RESUMO Introdução: Camphenol Plus é um derivado clorofenólico utilizado como medicação intracanal durante tratamentos pulporradiculares em Estomatologia. Existem poucos relatos científicos sobre o papel dos canais iônicos de potássio na dinâmica contrátil do músculo liso arterial induzida pela referida droga. Objetivo: Determinar o papel dos canais iônicos de potássio na dinâmica contrátil do músculo liso arterial induzida por Camphenol Plus. Método: Uma investigação experimental pré-clínica foi realizada no Instituto de Fisiologia "Oscar Langerdorff" da Faculdade de Medicina da Universidade de Rostock, Alemanha, entre outubro e dezembro de 2018, utilizando 30 anéis aórticos obtidos de 10 ratos Wistar (n=10). As preparações biológicas foram colocadas em banho de órgãos e pré-ativadas com solução de Krebs concentrada em íons potássio, registrando-se então a tensão desenvolvida pelo músculo liso vascular após a adição de soluções de Camphenol Plus em diferentes intervalos de tempo. Foram utilizados os testes U de Wilcoxon e Mann-Whitney. Resultados: 31,4% da musculatura lisa vascular relaxada pela ação do Camphenol Plus após pré-ativação com solução de Krebs concentrada em íons potássio. A maior diminuição do tônus vascular ocorreu com o uso de soluções medicamentosas a 7% entre o primeiro e o terceiro minutos. Conclusões: O efeito vasorrelaxante in vitro produzido pelo Camphenol Plus no músculo liso arterial é mediado por canais de íons de potássio sensíveis à voltagem, trifosfato de cálcio e adenosina do endotélio vascular e do sarcolema.

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RESUMEN Introducción: el canfenol plus, es un derivado clorado del fenol empleado de forma habitual como medicación intraconducto durante los tratamientos pulporradiculares en Estomatología. Son escasos los estudios en relación con sus efectos sobre el músculo liso vascular arterial y la participación del endotelio en estos. Objetivo: determinar la dependencia endotelial del efecto de canfenol plus 3 % sobre el músculo liso vascular arterial. Material y Métodos: se realizó una investigación experimental preclínica utilizando 26 anillos de carótida externa desprovistos de endotelio vascular. Las preparaciones realizadas se colocaron en baño de órganos, registrándose la tensión desarrollada por el músculo liso vascular tras la adición de acetilcolina, así como de soluciones de canfenol plus 3 % durante diferentes intervalos de tiempo. La dependencia entre ambas tensiones, se determinó a través de un modelo de regresión lineal simple. Resultados: tras la preactivación con solución Krebs concentrada de iones potasio, la adición de 10 μl de acetilcolina y canfenol plus 3 %, indujeron una discreta, pero significativa vasorrelajación de la musculatura lisa vascular. El modelo de regresión lineal elaborado, demostró la dependencia entre las variables tensión producida por acetilcolina y tensión producida por el fármaco al décimo minuto, corroborando la implicación del endotelio vascular en dicho efecto relajante. Conclusiones: el canfenol plus 3 %, produjo in vitro, un efecto vasorrelajante sobre la musculatura lisa de anillos de carótida externa, dependiente de endotelio y a partir de un factor relajante o hiperpolarizante derivado de este.

ABSTRACT Introduction: Camphenol plus is a chlorinated phenol derivative commonly used as intracanal medication during pulporradicular treatments in Dentistry. Studies in relation to its effects on arterial vascular smooth muscle and the involvement of the endothelium in them are scarce. Objective: To determine the endothelial dependence of the effect of 3 % camphenol plus on arterial vascular smooth muscle. Material and Methods: A preclinical experimental research was carried out using 26 external carotid artery rings devoid of vascular endothelium. The preparations made were placed in an organ bath, recording the tension developed by the vascular smooth muscle after the addition of acetylcholine, as well as 3 % Camphenol plus solutions during different intervals of time. The dependence between both tensions was determined through a simple linear regression model. Results: After pre-activation with Krebs concentrated potassium ion solution, the addition of 10 μl of acetylcholine and 3 % camphenol plus induced a discrete but significant vasorelaxation of the vascular smooth muscle. The linear regression model developed demonstrated the dependence between the variables tension produced by acetylcholine and tension produced by the drug at the tenth minute, corroborating the involvement of the vascular endothelium in that vasorelaxant effect. Conclusions: It is concluded that 3 % Camphenol plus in vitro, produced a vasorelaxant effect on the smooth muscle of external carotid rings dependent on endothelium and from a relaxing or hiperpolarizing factor derived from it.

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Resumen: Introducción: Las células de la musculatura lisa vascular (CMLV) se caracterizan por mantener cierto grado de desdiferenciación, variando su fenotipo entre el contráctil y el secretor, de acuerdo con las necesidades del tejido, y el contráctil predominante en condiciones fisiológicas. Cualquier alteración del estímulo mecánico, ya sea en el flujo sanguíneo o la tensión mecánica ejercida sobre las CMLV, conducen a cambios de su fenotipo y remodelamiento de la vasculatura, lo que puede constituir el punto de inflexión de varias patologías relevantes en la salud pública como, por ejemplo, la hipertensión arterial. Objetivo: Realizar una revisión sobre los mecanosensores y las vías transduccionales conocidas e implicadas en el cambio de fenotipo de las CMLV. Metodología: Se realizó una búsqueda sistemática en las bases de datos PubMed, Scopus, Google Académico y Scielo sobre la mantención y cambio de fenotipo de las células de la musculatura lisa vascular asociado principalmente a el estrés mecánico, la participación de los mecanosensores más relevantes y las vías de señalización involucrados en este proceso. Conclusión: Los mecanosensores implicados en el cambio de fenotipo de las CMLV contemplan principalmente receptores acoplados a proteína G, moléculas de adhesión y canales iónicos activados por estiramiento. Los estudios se han concentrado en la activación o inhibición de vías como las proteínas quinasas activadas por mitógenos (MAPK), la vía AKT, mTOR y factores transcripcionales que regulan la expresión de genes de diferenciación y/o desdiferenciación, como las miocardinas. Existen además otros receptores involucrados en la respuesta al estrés mecánico, como los receptores tirosina quinasas. A pesar de la importancia que reviste el conocimiento de los mecanosensores y las vías implicadas en el cambio de fenotipo de las CMLV, así como el papel que cumplen en el establecimiento de patologías vasculares, es aún escaso el conocimiento que se tiene sobre los mismos.

Abstract: Introduction: Vascular smooth muscle cells (VS- MCs) are characterized by maintaining a certain de- gree of dedifferentiation. VSMCs may vary their phenotype between contractile and secretory according to tissue needs. Under physiological conditions, the predominant phenotype is contractile. Any alteration of the mechanical stimulus, either in the blood flow or the mechanical stress exerted on the VSMCs, leads to changes in their phenotype and remodeling of the vasculature. These changes can constitute the turning point in several hypertension and other diseases relevant in public health. Objective: To review the main mechanosensor and transduction pathways involved changes in VSMCs phenotype. Methods: A systematic search of PubMed, Scopus, Google Scholar and Scielo databases was carried out to ascertain the state of the art regarding the maintenance and change of VSMCs phenotype mainly associated with mechanical stress. Additionally, the participation of the most relevant mechanosensors and the signaling pathways involved in this process are discussed. Conclusion: The mechanosensors involved in the change in VSMCs phenotype mainly contempla- te G-protein-coupled receptors, adhesion molecules, and stretch-activated ion channels. Studies have been focused on the activation or inhibition of MAPK, AKT, mTOR, pathways and transcriptional factors that regulate the expression of differentiation and/or des differentiation genes such as Myocardins. There are also other receptors involved in the response to mechanical stress such as the tyrosine kinases receptor. Although the importance of understanding mechanosensors, the signaling pathways involved in VSMC phenotype switching and their role in the establishment of vascular pathologies, knowledge about them is limited.

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Little is known about the effects of training load on exercise-induced plasma increase of interleukin-6 (IL-6) and soluble IL-6 receptor (sIL-6R) and their relationship with vascular remodeling. We sought to evaluate the role of sIL 6R as a regulator of IL-6-induced vascular remodeling. Forty-four male marathon runners were recruited and allocated into two groups: low-training (LT, <100 km/week) and high-training (HT, ≥100 km/week), 22 athletes per group. Twenty-one sedentary participants were used as reference. IL-6, sIL-6R and sgp130 levels were measured in plasma samples obtained before and immediately after finishing a marathon (42.2-km). Aortic diameter was measured by echocardiography. The inhibitory effect of sIL-6R on IL-6-induced VSMC migration was assessed using cultured A7r5 VSMCs. Basal plasma IL-6 and sIL-6R levels were similar among sedentary and athlete groups. Plasma IL-6 and sIL-6R levels were elevated after the marathon, and HT athletes had higher post-race plasma sIL-6R, but not IL-6, level than LT athletes. No changes in sgp130 plasma levels were found in LT and HT groups before and after running the marathon. Athletes had a more dilated ascending aorta and aortic root than sedentary participants with no differences between HT and LT athletes. However, a positive correlation between ascending aorta diameter and plasma IL-6 levels corrected by training load and years of training was observed. IL-6 could be responsible for aorta dilation because IL-6 stimulated VSMC migration in vitro, an effect that is inhibited by sIL-6R. However, IL-6 did not modify cell proliferation, collagen type I and contractile protein of VSMC. Our results suggest that exercise induces vascular remodeling. A possible association with IL-6 is proposed. Because sIL-6R inhibits IL-6-induced VSMC migration, a possible mechanism to regulate IL-6-dependent VSMC migration is also proposed.

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Introducción: Uno de los derivados de los clorofenoles más utilizado en Estomatología, lo constituye el p-clorofenol (4-clorofenol), empleado como agente antibacteriano en la desinfección del conducto radicular durante el tratamiento pulporradicular. Son escasos los reportes científicos sobre sus efectos en la musculatura lisa vascular arterial y la regulación del flujo sanguíneo local. Objetivo: Determinar el efecto del 4-clorofenol sobre el músculo liso vascular de aorta abdominal de ratas Wistar. Material y Métodos: Se realizó una investigación experimental preclínica, utilizando 30 anillos de aorta abdominal (porción superior) obtenidos de ratas Wistar adultas. Las preparaciones de unos 5 mm se colocaron en baño de órganos, registrándose la tensión desarrollada por el músculo liso vascular tras la adición de 4-clorofenol en diferentes concentraciones y durante diferentes intervalos de tiempo. Resultados: El 4-clorofenol, tras la preactivación del musculo liso vascular de anillos de aorta abdominal, indujo relajación del vaso, la que se incrementó durante todo el tiempo de estudio y al aumento de la concentración del medicamento. Existieron diferencias significativas entre los valores de tensión promedios registrados en los diferentes intervalos de tiempo con los de la tensión base inicial. Conclusiones: El p-clorofenol indujo in vitro, relajación del músculo liso vascular de aorta abdominal de ratas Wistar(AU)

Introduction: In Dentistry, p-chlorophenol (4-chlorophenol) is one of the most widely used derivatives of chlorophenols. It is used as an antibacterial agent in root canal disinfection during pulp-radicular treatment. There are few scientific reports on its effects on vascular smooth musculature and the regulation of local blood flow. Objective: To determine the effect of 4-chlorophenol on vascular smooth muscle of abdominal aorta from Wistar rats. Material and Methods: A preclinical experimental research was carried out using 30 abdominal aortic rings (upper portion) obtained from adult Wistar rats. The preparations of about 5 mm were placed in an organ bath, recording the tension developed by the vascular smooth muscle after the addition of 4-chlorophenol at different concentrations and during different time intervals. Results: The results demonstrate that 4-Chlorophenol induced vasorelaxation after the preactivation of the vascular smooth muscle of the abdominal aortic rings, which increased during the entire study time and with increased drug concentration. There were significant differences among average tension values registered at different intervals of time in relation to the initial base tension. Conclusions: It is concluded that in vitro, p-chlorophenol induced relaxation of abdominal aorta vascular smooth muscle in Wistar rats(AU)

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