Intact Arteries Research Articles

Control of Vasoconstriction by Myoendothelial Ca2+ Signaling Through Gap Junctions

In resistance arteries, smooth muscle cells (SMCs) and endothelial cells (ECs) are communicated through gap junctions (i.e. myoendothelial gap junctions), which provide a signaling pathway for control of vasomotor tone. In this context, the Ca2+ signaling associated to smooth muscle contraction might also be transmitted via myoendothelial gap junctions and activate a regulatory feedback mechanism (myoendothelial feedback) by the production of endothelium‐dependent vasodilators. It is thought that IP3 is the main Ca2+‐related signaling transmitted from smooth muscle cells to endothelial cells, but the direct contribution of Ca2+ to the myoendothelial feedback is not clear. We investigated the temporal pattern of vasoconstriction in mesenteric resistance arteries, and their temporal relationship with the increase of Ca2+ in SMC and EC. We used intact mesenteric resistance arteries of Sprague dawley male rats (≤ 10 mm in length) to measure the vasoconstriction and changes in [Ca2+]i of smooth muscle cells and endothelial cells observed in response to phenylephrine (PE) or KCl using the fluorescent indicators of Ca2+, Fluo‐4 and X‐Rhod‐1.Changes in [Ca2+]i were also evaluated in primary cultures of mesenteric endothelial cells. All procedures were approved by the local Bioethical Committee (ID:180806005). The vasoconstriction induced by PE and KCl was not only associated with an increase of [Ca2+]i in smooth muscle cells, but also in endothelial cells, which was prevented by the blockade of L‐type voltage‐dependent Ca2+ channels with 10 μM nifedipine or the inhibition of gap junctions with 50 μM 18‐b‐Glycyrrhetenic acid. Although the stimulation with PE or KCl did not evoke an increment of [Ca2+]i in primary cultures of endothelial cells, the response induced by these vasoconstrictors was larger after blocking nitric oxide (NO) production with NG‐nitro‐L‐arginine (100 μM). These results suggest that the direct diffusion of Ca2+ contributes to the NO‐mediated component of the myoendothelial feedback.Support or Funding InformationConicyt 21171840

Exercise‐mediated Wall Shear Stress Attenuates NLRP3 Inflammasome‐induced Endothelial Dysfunction in Obesity

PURPOSENLRP3 inflammasome plays a pivotal role in obesity‐induced vascular pathology through caspase‐1‐mediated IL‐1β processing, while precise mechanism by which exercise ameliorates the inflammasome remains unknown. Thus, we investigated the effect of exercise on endothelial dysfunction in obesity via NLRP3 inflammasome‐dependent mechanisms.METHODSC57BL/6J male mice were provided a control low‐fat diet (LFD; 4.5% calories from fat) or a high‐fat diet (HFD; 45% calories from fat) with a running wheel access (LF‐RUN or HF‐RUN) or a sedentary no‐wheel condition (LF‐SED or HF‐SED) for 12 to 14 weeks. Endothelium‐dependent vasodilation in response to increasing acetylcholine (ACh, 1 nmol/L to 10 μmol/L) was assessed using intact mesentery arteries isolated from mice. Mouse microvascular endothelial cell (MVEC) line EOMA was treated with palmitate (PA, 20 μmol/L) and laminar shear stress (LSS, 20 dynes/cm2): Static control (STT), PA, LSS, and LSS+PA. The NLRP3 inflammasome axis, including NLRP3, ASC, caspase‐1, p20, and IL‐1β in mice aorta and MVECs was examined by RT‐qPCR, FLICA and DAB staining and western blot.RESULTSHFD significantly increased obesity‐associated parameters while these were attenuated by running exercise. Obesity greatly decreased ACh‐induced endothelium‐dependent vasodilation compared to LF‐SED. Exercise improved reduced vasodilator response to ACh in obese mice. Incubation of caspase‐1 inhibitor (10 μmol/L) to the isolated vessel restored obesity‐induced impairment of vasodilator response similar to one of LF‐SED, LF‐RUN, and HF‐RUN. Exercise‐induced decrease of caspase‐1 and IL‐1β in endothelial cells of obese mice aorta was determined by immunohistochemistry using FLICA and DAB, respectively. Likewise, in in vitro experiment, LSS diminished inflammasome components in PA‐treated MVECs.CONCLUSIONThese findings suggest that exercise attenuates against obesity‐induced endothelial dysfunction through a shear stress‐dependent mechanism. Our finding suggests that exercise may be a therapeutic strategy for inflammation‐related vascular dysfunction in obesity.Exercise attenuates IL‐1β in endothelium of obese mice aorta.Immunohistochemical analysis of IL‐1β expression (dark brown precipitate) in the endothelial layer of the aorta. Summarized data showing area percentage of the endothelium positive for IL‐1β in aorta (n=4–6). Values are means ± SEM. * P<.05 vs. LFSED; # P<.05 vs. HF‐SED.Figure 1Inhibition of caspase‐1 abolishes endothelial dysfunction in mesentery arteries of obese mice.Incubation of the caspase‐1 inhibitor, Ac‐YVAD‐cmk, in isolated mesentery arteries from obese mice showing improved ACh‐induced endothelium‐dependent vasodilation in HF‐SED + Ac‐YVAD‐cmk (n = 3) similar to the levels of LF‐SED (n=8), HF‐RUN (n = 9) and HF‐RUN + Ac‐YVAD‐cmk (n = 7). Values are means ± SEM. ## P<.01 vs. HF‐SED.Figure 2

Giant \u201cheart appearance-like sign\u201d on MRI in bilateral ponto-medullary junction infraction: case report

BackgroundBilateral medial medullary infarction (MMI) is uncommon and bilateral medial pons infarction (MPI) is even rarer. “Heart appearance” on magnetic resonance imaging (MRI) is a characteristic presentation of bilateral medial medullary infarction (MMI).Case presentationWe present 67-year-old Chinese diabetic and hypertensive female patient affected with “heart appearance-like” infarction in bilateral ponto-medullary junction on MRI. Abnormal signal was observed in the bilateral ponto-medullary junction on T1, T2, fluid-attenuated inversion recovery and apparent diffusion coefficient (ADC). The whole brain digital subtraction angiography (DSA) showed the basilar artery and vertebral artery remained intact. Therefore, we speculated that the bilateral ponto-medullary junction infarction might be caused by the deep perforating branch of the basilar artery.ConclusionsAs far as we know, the “heart appearance-like” infraction in bilateral ponto-medullary junction was not reported. Our case also suggests that bilateral ischemic infraction involvement of the medulla and pon is possible even in the context of an intact basilar artery.

Prediction of Subclinical Coronary Atherosclerosis in Patients with High and Very High Cardiovascular Risk

Objective To develop a diagnostic rule for detection of patients (pts) with high probability of subclinical atherosclerosis among those with high or very high cardiovascular (CV) risk.Materials and Methods This cross-sectional study enrolled 52 pts (32 men [62 %]), aged 40 to 65 years [mean age 54.6±8.0]) with high or very high CV risk (5-9 and ≥10 % by The Systematic Coronary Risk Estimation Scale [SCORE], respectively). All participants underwent cardiac computed tomography (CT) angiography and calcium scoring. Traditional risk factors (RFs) (family history of premature CVD, smoking, overweight / obesity and abdominal obesity, hypertension, type 2 diabetes mellitus, lipids parameters (total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides) and lipids-related markers (apolipoprotein A1, apolipoprotein B, ApoB / ApoA1 ratio), biomarkers of inflammation (high-sensitivity C-reactive protein [hs CRP], fibrinogen), indicator carbohydrate metabolism (glucose), ankle-brachial index, stress-test, carotid plaques according to ultrasound were evaluated in all pts. Psychological RFs were evaluated using Hospital Anxiety and Depression Scale and DS-14 for type D personality.Results All pts were divided into 2 groups according to the CT angiography results: pts in the main group (n=21) had any non-obstructive lesions or calcium score >0, pts in the control group (n=31) had intact coronary arteries. The groups did not differ in age or gender. 26 multiple linear logistic models for any subclinical atherosclerosis were developed based on obtained diagnostic features. Taking into account R-square = 0.344 (p=0.0008), the best fitting model was follows: subclinical coronary atherosclerosis= -1.576 + 0.234 x SCORE ≥5 % + 0.541 x hs CRP >2 g / l +0.015 x heart rate (bpm) +0.311 family history of premature CVD. The developed algorithm had sensitivity of 63 % and specificity of 80 %.Conclusion The created diagnostic model diagnostic model suggests the presence of subclinical coronary atherosclerosis in patients with high / very high CV risk with a high degree of probability. This easy-to-use method can be used in routine clinical practice to improve risk stratification and management choices in high-risk pts.

Decision-making and therapeutic options in intact splenic artery aneurysms: single-center experience and literature review.

Splenic artery aneurysms are rare, potentially serious, and usually asymptomatic. Several methods are currently available to treat them, each with their own advantages and drawbacks. Therefore, its therapeutic paradigm has changed. We review our database of splenic aneurysms (2009-2019) and undertake an exhaustive literature review. Demographic, clinical, diagnostic, therapeutic, early and follow-up outcome data were examined. Our experience comprised: 15 patients with 19 splenic aneurysms. 11 women (average age, 59.4 years) and 4 men (average age, 61.7 years). All asymptomatic. At diagnosis, aneurysms had a mean cross-sectional diameter of 3.4 cm (3.2 and 3.9 for women and men, respectively), the largest measuring 8.5 cm. Two independent aneurysms were detected in four patients. Diagnoses were always incidental to a CT scan. Treatments consisted of open surgery (2 patients), endovascular surgery (10 patients: 7 embolizations, 3 covered stent) and observation/follow-up (3 patients). The cases of open surgery (with splenectomy) were carried out without postoperative morbidity. One embolization failed (requiring subsequent open surgery) and two suffered localized splenic infarction, but without further complications. In patients treated with a covered stent, the aneurysm was always excluded, without complications. There was no 30-day or follow-up (average 26.2 months) mortality. Splenic aneurysms are diagnosed more frequently and earlier (in the asymptomatic phase), albeit incidentally, than in the past. The correct indication (identifying patients at risk) and individualization of treatment, in which endovascular techniques are the first-line option, have significantly improved morbidity and mortality outcomes in our hospital.

FK506 regulates Ca2+ release evoked by inositol 1,4,5-trisphosphate independently of FK-binding protein in endothelial cells.

Background and PurposeFK506 and rapamycin are modulators of FK‐binding proteins (FKBP) that are used to suppress immune function after organ and hematopoietic stem cell transplantations. The drugs share the unwanted side‐effect of evoking hypertension that is associated with reduced endothelial function and nitric oxide production. The underlying mechanisms are not understood. FKBP may regulate IP3 receptors (IP3R) and ryanodine receptors (RyR) to alter Ca2+ signalling in endothelial cells.Experimental ApproachWe investigated the effects of FK506 and rapamycin on Ca2+ release via IP3R and RyR in hundreds of endothelial cells, using the indicator Cal‐520, in intact mesenteric arteries from male Sprague‐Dawley rats. IP3Rs were activated by acetylcholine or localised photo‐uncaging of IP3, and RyR by caffeine.Key ResultsWhile FKBPs were present, FKBP modulation with rapamycin did not alter IP3‐evoked Ca2+ release. Conversely, FK506, which modulates FKBP and blocks calcineurin, increased IP3‐evoked Ca2+ release. Inhibition of calcineurin (okadiac acid or cypermethrin) also increased IP3‐evoked Ca2+ release and blocked FK506 effects. When calcineurin was inhibited, FK506 reduced IP3‐evoked Ca2+ release. These findings suggest that IP3‐evoked Ca2+ release is not modulated by FKBP, but by FK506‐mediated calcineurin inhibition. The RyR modulators caffeine and ryanodine failed to alter Ca2+ signalling suggesting that RyR is not functional in native endothelium.Conclusion and ImplicationsThe hypertensive effects of the immunosuppressant drugs FK506 and rapamycin, while mediated by endothelial cells, do not appear to be exerted at the documented cellular targets of Ca2+ release and altered FKBP binding to IP3 and RyR.

Effects of intrauterine growth restriction on Ca2+-activated force and contractile protein expression in the mesenteric artery of 1-year-old Wistar-Kyoto rats

Intrauterine growth restriction (IUGR) affects vascular reactivity in older rats, but at present the causative factors for this change are unknown. Therefore, we investigated downstream events associated with vascular reactivity, specifically, Ca2+-regulated force production and shifts in contractile protein content. The mesenteric artery from male and female 1-year-old Wistar-Kyoto rats was examined using two distinct experimental growth restriction models. Uterine ligation surgery restriction or a sham surgery was conducted at day 18 of pregnancy, whilst a food restriction diet (40% control diet) began on gestational day 15. Extracellular vascular reactivity was studied using intact mesenteric arteries, which were subsequently chemically permeabilized using 50μM β-escin to examine Ca2+-activated force. Peak contractile responses to a K+-induced depolarization and phenylephrine were significantly elevated due to an increase in maximum Ca2+-activated force in the male surgery restricted group. No changes in contractile forces were reported between female experimental groups. Sections of mesenteric artery were examined using western blotting, revealing IUGR increased the relative abundance of the voltage-gated Ca2+ channel, inositol-1,4,5-trisphosphate receptor and myosin light chain kinase, in both male growth restricted groups, whereas no changes were seen in females. These findings demonstrate for the first time in 1-year-old rats that changes in vascular reactivity due to IUGR are caused by a change in Ca2+-activated force and shifts in important contractile protein content. These changes affect the Wistar-Kyoto rat in a sex-specific and maternal insult-dependent manner.

Epac-mediated relaxation in murine basilar arteries depends on membrane permeability of cyclic nucleotide analogues and endothelial aging

Cerebral blood supply is finely tuned by regulatory mechanisms depending on vessel caliber the disruption of which contributes to the development of diseases such as vascular dementia, Alzheimer's and Parkinson 's diseases. This study scopes whether cAMP-mimetic-ligands relax young and aged murine cerebral arteries, whether this relates to the activation of PKA or Epac signaling pathways and is changed with advanced age. The hormone Urocortin-1 relaxed submaximally contracted young and old basilar arteries with a similar pD2 and DMAX (~ -8.5 and ~ 90% in both groups). In permeabilized arteries, PKA activation by 6-Bnz-cAMP or Epac activation by 8-pCPT-2'- O-Me-cAMP also induced relaxation with pD2 of -6.3 vs. -5.8 in old for PKA-ligands, and -4.4 and -4.0 in old for Epac-ligands. Furthermore, aging significantly increased submaximal Ca2+-induced force. The effect of 8-pCPT-2'-O-Me-cAMP on intact arteries was attenuated by aging or nitric oxide synthase inhibition. No relaxing effect in both age-groups was observed after treatment with PKAactivator, Sp-6-Phe-cAMPS. In conclusion, our results suggest that in intact basilar arteries relaxation induced by cAMP-mimetics refers only to the activation of Epac and is impaired by smooth muscle and endothelial aging. The study presents an interesting option allowing therapeutic discrimination between both pathways, possibly for the exclusive activation of Epac in brain circulatory system.

Biochemical features of blood vessel extracellular calcium-sensing receptor and their physiological mechanism of action in the regulation of vascular tone and blood pressure

The extracellular calcium-sensing receptor (CaR) is a G protein coupled receptor superfamily that has ability to sense very small changes in extracellular Ca2+ with primary physiological function of maintenance of constant blood Ca2+ levels. In addition to being expressed in tissues involved in [Ca2+]o homeostasis, it is also expressed in tissues not involved in mineral homeostasis, suggestive of additional physiological functions such as control vasomotor tone and blood pressure. However, the exact cellular mechanisms involved in these responses remain unclear. Stimulation of CaRs on perivascular nerves is proposed to evoke synthesis and release of nitric oxide (NO) from pre-synaptic terminals, which activate large conductance Ca2+-activated K+ channels in adjacent VSMCs that induce membrane hyperpolarisation and subsequent vasorelaxation. Plus, activation of CaRs on endothelial cells is reported to generate NO or vasoactive lipids or induce activation of intermediate conductance Ca2+-activated K+ channels to induce vasorelaxations. In contrast, CaR-induced activation of Gq and MAPK kinase-mediated pathways in VSMCs are linked to vasoconstriction and cell proliferation respectively. Taken together, these findings indicate that stimulation of vascular CaRs is reported to induce both constrictions and relaxations. In the endothelium intact artery, CaR mediated vasorelaxations are the dominant physiological phenomenon.

An exploration of potential approaches to improve the diagnosis of subclinical atherosclerosis in patients with high cardiovascular risk

The search for optimal approaches to the diagnosis of subclinical atherosclerosis using a wide range of traditional and psychosocial risk factors (RFs), as well as clinical and instrumental diagnostic methods in patients (pts) with high or very high cardiovascular (CV) risk. This cross-sectional study enrolled52 pts, aged 40 to 65 years with high or very high CV risk (5-9 and ≥10% by the Systematic Coronary Risk Estimation Scale [SCORE], respectively). All participants underwent cardiac computed tomography (CT)angiography and calcium scoring. Traditional RFs (family history of premature CVD, smoking, overweight/obesity and abdominal obesity, hypertension, type 2 diabetes mellitus, lipids parameters (total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides) and lipids-related markers (apolipoprotein A1, apolipoprotein B, ApoB/ApoA1 ratio), biomarkers of inflammation (high-sensitivity C-reactive protein [hs CRP], fibrinogen), indicator carbohydrate metabolism (glucose), ankle-brachial index, stress-test, carotid plaques according to ultrasound, arterial stiffness were evaluated in all pts. Psychological RFs were evaluated using Hospital Anxiety and Depression Scale and DS-14 for type D personality. All pts were divided into 2 groups according to the CT angiography results: pts in the main group (n=21) had any non-obstructive lesions or calcium score >0, pts in the control group (n=31) had intact coronary arteries. The groups did not differ in age or gender. It was found that patients with subclinical atherosclerosis significantly more often have a very high (≥10%) CV risk (42.9% vs.16.3%, p<0.05), a long (≥5 years) history of arterial hypertension (47.6% vs. 12.9% , p<0.01) and longer duration of antihypertensive therapy (61.9% vs. 29.0%, p<0.05), higher heart rate in rest (87. ± 14 vs. 77 ± 10 beats/min, p<0.01), increased arterial stiffness according to aortic pulse wave velocity (85.7% vs. 61.3%, p<0.05) and high level of hs-CRP (100% vs. 90.3%, p<0.05). Using in routine clinical practice of additional anamnestic (hypertension lasting ≥ 5 years and the intake of any antihypertensive drugs) and clinical-instrumental parameters (high heart rate in rest, hs CRP and arterial stiffness in pts with high and very high CV risk increases effectiveness of early detection of subclinical atherosclerosis.

Long-Term Outcomes of Digital Nerve Repair Accompanied by Digital Artery Injury in Flexor Zone 2.

Objectives Better healing results of any tissue or area is closely linked with a well-blood supply in reconstructive surgery. Peripheric nerve healing is closely related to blood supply as well. We aimed to assess whether there was any difference between digital nerve healing with and without extrinsic blood supply. Methods We assessed 48 patients with unilateral digital nerve injury at zone 2. Twenty-four of them had unrepairable arterial injury and other 24 had no arterial injury. The 24 patients in the “unrepaired artery group” (UA) and 24 patients in the “intact artery group” (IA) were compared. Results Mean follow-up time was 17.7 months. The mean two-point discrimination (2PD) was 5.29 mm in IA group and 5.37 mm in UA group. One neuroma in IA group and two neuromas in UA group were determined. We found no statistically significant difference between these groups in terms of neuroma, 2PD, and cold intolerance. The results of British Medical Research Council sensory recovery clinical scale were comparable for these two groups. Conclusion Digital nerve healing is related to numerous factors. We hypothesized that blood flow may be one of these factors; however, at this zone digital artery repair is not the foremost determinant for digital nerve healing. Further researches should be done for upper injury levels. Despite this result, we argue not to leave the digital artery without repairment and we propose to repair both artery and nerve to achieve the normal anatomical integrity and to warrant finger blood flow in possible future injuries.

Calcium Phosphate Bions Cause Intimal Hyperplasia in Intact Aortas of Normolipidemic Rats through Endothelial Injury.

Calcium phosphate bions (CPBs) are formed under blood supersaturation with calcium and phosphate owing to the mineral chaperone fetuin-A and representing mineralo-organic particles consisting of bioapatite and multiple serum proteins. While protecting the arteries from a rapid medial calcification, CPBs cause endothelial injury and aggravate intimal hyperplasia in balloon-injured rat aortas. Here, we asked whether CPBs induce intimal hyperplasia in intact rat arteries in the absence of cardiovascular risk factors. Normolipidemic Wistar rats were subjected to regular (once/thrice per week over 5 weeks) tail vein injections of either spherical (CPB-S) or needle-shaped CPBs (CPB-N), magnesium phosphate bions (MPBs), or physiological saline (n = 5 per group). Neointima was revealed in 3/10 and 4/10 rats which received CPB-S or CPB-N, respectively, regardless of the injection regimen or blood flow pattern in the aortic segments. In contrast, none of the rats treated with MPBs or physiological saline had intimal hyperplasia. The animals also did not display signs of liver or spleen injury as well as extraskeletal calcium deposits. Serum alanine/aspartate transaminases, interleukin-1β, MCP-1/CCL2, C-reactive protein, and ceruloplasmin levels did not differ among the groups. Hence, CPBs may provoke intimal hyperplasia via direct endothelial injury regardless of their shape or type of blood flow.

Hydrogen peroxide depolarizes mitochondria and inhibits IP3-evoked Ca2+ release in the endothelium of intact arteries

Hydrogen peroxide (H2O2) is a mitochondrial-derived reactive oxygen species (ROS) that regulates vascular signalling transduction, vasocontraction and vasodilation. Although the physiological role of ROS in endothelial cells is acknowledged, the mechanisms underlying H2O2 regulation of signalling in native, fully-differentiated endothelial cells is unresolved. In the present study, the effects of H2O2 on Ca2+ signalling were investigated in the endothelium of intact rat mesenteric arteries. Spontaneous local Ca2+ signals and acetylcholine evoked Ca2+ increases were inhibited by H2O2. H2O2 inhibition of acetylcholine-evoked Ca2+ signals was reversed by catalase. H2O2 exerts its inhibition on the IP3 receptor as Ca2+ release evoked by photolysis of caged IP3 was supressed by H2O2. H2O2 suppression of IP3-evoked Ca2+ signalling may be mediated by mitochondria. H2O2 depolarized mitochondria membrane potential. Acetylcholine-evoked Ca2+ release was inhibited by depolarisation of the mitochondrial membrane potential by the uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP) or complex 1 inhibitor, rotenone. We propose that the suppression of IP3-evoked Ca2+ release by H2O2 arises from the decrease in mitochondrial membrane potential. These results suggest that mitochondria may protect themselves against Ca2+ overload during IP3-linked Ca2+ signals by a H2O2 mediated negative feedback depolarization of the organelle and inhibition of IP3-evoked Ca2+ release.

Extracellular vesicle integrins act as a nexus for platelet adhesion in cerebral microvessels

Circulating extracellular vesicles (EVs) regulate signaling pathways via receptor-ligand interactions and content delivery, after attachment or internalization by endothelial cells. However, they originate from diverse cell populations and are heterogeneous in composition. To determine the effects of specific surface molecules, the use of synthetic EV mimetics permits the study of specific EV receptor-ligand interactions. Here, we used endogenous EVs derived from the circulation of rats, as well as ligand-decorated synthetic microparticles (MPs) to examine the role of integrin αvβ3 in platelet adhesion under flow in structurally intact cerebral arteries. At an intraluminal pressure of 50 mmHg and flow rate of 10 µl/min, platelets were delivered to the artery lumen and imaged with whole-field fluorescent microscopy. Under basal conditions very few platelets bound to the endothelium. However, adhesion events were markedly increased following the introduction of arginine-glycine-aspartate (RGD)-labelled synthetic MPs or endogenously-derived EVs from experimental stroke animals carrying excess RGD proteins, including vitronectin, CD40-ligand and thrombospondin-1. These data, which were generated in a dynamic and physiologically relevant system, demonstrate the importance of vesicle-carried RGD ligands in platelet adherence to the cerebrovascular endothelium and highlight the ability of synthetic EVs to isolate and identify key components of the molecular handshake between EVs and their targets.

"A Step and a Ceiling": mechanical properties of Ca2+ spark vasoregulation in resistance arteries by pressure-induced oxidative activation of PKG.

We investigated the biomechanical relationship between intraluminal pressure within small mesenteric resistance arteries, oxidant activation of PKG, Ca2+ sparks, and BK channel vasoregulation. Mesenteric resistance arteries from wild type (WT) and genetically modified mice with PKG resistance to oxidative activation were studied using wire and pressure myography. Ca2+ sparks and Ca2+ transients within vascular smooth muscle cells of intact arteries were characterized using high‐speed confocal microscopy of intact arteries. Arteries were studied under conditions of varying intraluminal pressure and oxidation. Intraluminal pressure specifically, rather than the generic stretch of the artery, was necessary to activate the oxidative pathway. We demonstrated a graded step activation profile for the generation of Ca2+ sparks and also a functional “ceiling” for this pressure –‐sensitive oxidative pathway. During steady state pressure ‐ induced constriction, any additional Ca2+ sensitive‐K+ channel functional availability was independent of oxidant activated PKG. There was an increase in the amplitude, but not the Area under the Curve (AUC) of the caffeine‐induced Ca2+ transient in pressurized arteries from mice with oxidant‐resistant PKG compared with wild type. Overall, we surmise that intraluminal pressure within resistance arteries controls Ca2+ spark vasoregulation through a tightly controlled pathway with a graded onset switch. The pathway, underpinned by oxidant activation of PKG, cannot be further boosted by additional pressure or oxidation once active. We propose that these restrictive characteristics of pressure‐induced Ca2+ spark vasoregulation confer stability for the artery in order to provide a constant flow independent of additional pressure fluctuations or exogenous oxidants.

Synthesis and evaluation of substituted 8,8-dimethyl-8H-pyrano[2,3-f]chromen-2-one derivatives as vasorelaxing agents

A series of substituted 8,8-dimethyl-8H-pyrano[2,3-f]chromen-2-ones (chromeno-coumarin hybrids) was synthesized from scopoletin (11) as vasorelaxing agents. The synthesized compounds 21a-f, 22, 23a-e and scopoletin (11) were evaluated for vasorelaxation in endothelium intact rat main mesenteric artery (MMA). Compounds 11, 21a, 21c-f and 22 showed significant vasorelaxation in precontracted MMA within the range of EC50 value 1.58–5.02 µM. These derivatives presented 29.40–70.89 fold increased sensitivity for experimental tissue compared to scopoletin (11), the parent molecule. Among others, 22 was found to be the most active compound which had EC50 1.58 µM with 70.89 fold increased sensitivity. The mechanistic evaluation of 22 showed that it exerted vasorelaxation through Ca2+-activated K+ (BKca) channel and the effect was endothelium-independent.

Hemostatic Gene Polymorphisms in Acute Coronary Syndrome with Nonobstructive Coronary Atherosclerosis

to study distribution of genes of the coagulation system, unfavorable in relation to the risk of thrombosis, and their influence on serum parameters ofthe hemostasis system in patients with nonobstructive coronary atherosclerosis (NCA) and acute coronary syndrome (ACS). We included in this nonrandomized open study patients with ACS older than 18 years with intact coronary arteries or confirmed at coronary angiography stenosis <50%. Genotypes of these patients were analyzed by 8 polymorphic variants of the hemostatic system genes which previously were found to be associated with the thrombophilia risk: F2 (20210 G>A) rs1799963, F5 (1691 G>A) rs6025, F7 (10976G>A) rs6046, F13 (163 G>T) rs5985, F1 (-455G>A) rs1800790, GP Ia - Ila (807C>t) rs1126643, GP Ilb-IIIa (1565 T>C) rs5918, PAI-I (-6755G>4G) rs1799889. Activities of protein C, Von Willebrand factor, plasminogen, and antithrombin III were also determined. Of 913 patients with ACS in 30 (3.3%) with mean age 54±11 years we detected NCA. Acute myocardial infarction (AMI) was diagnosed in 24 (80%), unstable angina - in 6 (20%) patients. Only in 1 patient we found no carriage of thrombosis associated genotypes. The frequency of occurrence of the heterozygous genotype of the factor V gene was 1 (3%). Heterozygous genotype of the factor XIII was registered significantly more often in patients with present atherosclerotic lesion compared with those with intact coronary arteries. Mean activity of protein C was 103% [90; 110], antithrombin III - 96% [88; 103], Von Willebrand factor - 137% [114; 162], plasminogen - 109% [102; 112]. At admission lowering of antithrombin III and protein C activities was detected in 4 cases (13%). In dynamics level of these parameters was restored. Elevation of Von Willebrand factor activity at admission was detected in 14 cases (14%) and remained elevated one year after the index event. There was no association between of fibrinogen level, protein C activity, rs1800790 and rs6025 gene polymorphisms, respectively. One-year mortality was 7% (n=2). For one year occurred 1 AIM recurrence (3%), heart failure developed in 15 patients (50%), 11 patients (37%) were repetitively hospitalized due to all causes. No association was revealed between activity of studied blood serum markers and 1 -year outcomes (death, re-AIM, rehospitalization). Among ACS patients 3.3% had NCA, what corresponded to the literature data. Carriage of at least 1 polymorphic variant of 8 thrombosis associated genes of the coagulation system was found in 97 % of patients with ACS and NCA. Distribution of these variants was like that in the European population and in patients with AIM at the background of stenosing atherosclerosis. Level of serum markers did not depend on distribution of polymorphic variants of the coagulation system genes, and presence of atherosclerotic coronary artery lesions. There was no association between hospital and long-term outcomes and distribution of polymorphic variants of thrombosis associated coagulation system genes, as well as levels of blood serum markers.

Internal carotid and vertebral artery dissection: morphology, pathophysiology and provoking factors

The causes of internal carotid artery (ICA) and vertebral artery (VA) dissection, as well as its provoking factors, remain understudied. The aim of this paper was to explore morphological changes in the ICA/VA walls, factors provoking dissection, clinical signs and biomarkers of connective tissue (CT) damage. A total of 271 patients were examined, of whom 54% were women. The mean age of the participants was 37.0 ± 10 years. Clinical signs and biomarkers of CT damage (matrix metalloproteinase 9, tissue inhibitor of metalloproteinase 1, hydroxyproline, sulphated glycosaminoglycans) were analyzed in 82 patients and 40 healthy volunteers. Histologic examination of dissected and seemingly intact arteries conducted in 5 cases revealed signs of arterial wall dysplasia similar to those characteristics of fibromuscular dysplasia: thinning and splitting of the internal elastic membrane, areas of fibrosis, irregular orientation of myocytes, and their necrosis in the tunica media. Clinical signs and biomarkers of CT dysplasia (CTD) were more pronounced in patients with arterial dissection than in the controls. The major provoking factors were head turns and physical activity (42%), minor head injury (10%), and acute respiratory infection in the month preceding arterial dissection (14%). We conclude that arterial wall dysplasia is a predisposing factor for ICA/VA dissection, both spontaneous and provoked. The analysis of CTD biomarkers and clinical signs suggests connective tissue pathology in patients with ICA/VA dissection.

P3596The role of MSCT and SPECT in patients with acute coronary syndromes and non-obstructive coronary artery disease

Abstract Background Approximately 1–14% of patients with ACS not have obstructive changes in the coronary arteries according to the data of invasive coronary angiography (ICA) is myocardial infarction (AMI) with non-obstructive coronary artery- MINOCA. MSCT has the ability to assess the lumen of the vessel, visualize the artery wall, the structure of the atherosclerotic plaque. SPECT is used to detect myocardial ischemia, determine the functional significance of atherosclerotic plaques, and evaluate regional tissue perfusion. There are no data on a research of a structurally functional condition of a coronary blood-groove by means of MSCT, SPECT.Aim. To study the role of MSCT and SPECT in patients with MINOCA, to compare MSCT with invasive coronarography. Material and methods The study is registered on ClinicalTrials.gov. Inclusion criteria are listed on the site. All patients underwent CT, SPECT, on the 6th day from the beginning of the index event. All MSCT and SPECT studies were performed on a 64-section combined single-photon-emission and X-ray computer tomograph GE Discovery NM/CT 570C. Endpoint: Frequency of occurrence of atherosclerosis, frequency of occurrence of unstable plaques according to MSCT, the magnitude of a transient defect in myocardial perfusion according to SPECT. Results The study included 14 patients with MINOCA, women predominate – 11 (78.6%), the average age was 61.1±14 years. The risk of GRACE was moderate in 8 (57%) pts. 12 (85.7%) pts were hospitalized within the first 6 hours of the onset of the disease.Thrombolytic therapy was performed in 3 (21.4%) pts, 2 of them were effective (14%). 57% of patients at admission were troponin-positive.According to the results of ICA, intact coronary arteries were detected in 9 (64.3%) pts, 5 (35%) had stenosis up to 50%. Coronary slow flow (TIMI 2) was determined in 11 (78.6%) pts, of which 8 (57%) pts have coronary slow flow and intact coronary arteries. Coronary spasm was in 1 (7.1%) patient. According to MSCT, the proportion of pts with intact coronary arteries decreased (9 (64.3%) → 5 (35.7%)) and the proportion with non-obstructive atherosclerosis increased (5 (35%) → 9 (64.3%)). All pts had a positive remodeling index, soft-tissue, predominantly calcified stenoses and eccentric plaques. The average Segment Involvement Score was 2.1 (0; 5), the Segment Stenosis Score was −2.4 (1; 3). 12 pts (85%) had transitory perfusion defects. The median SSS values were 7.5 (4; 13), SRS 4.7 (1.0; 9.0), SDS 4.7 (3.0; 8.0). Conclusion In 78% of pts had a slowing of the coronary blood flow and intact coronary arteries according to ICAG. However, according to MSCT, only 36% of the patients did not have plaques. MSCT allows to identify eccentric soft tissue atherosclerotic plaques that have a high risk of rupture/erosion, and, accordingly, the high risk of the development of AMI in this group. Using SPECT revealed a statistically significant transient perfusion defect.

P721Shear stress induced unfolding of von willebrand factor may be involved in the premature development of myocardial infarction

Abstract Background Normally, von Willebrand factor (vWF) becomes highly reactive with platelets upon unfolding into a fibrillar conformation at critical shear rate (more than 5000 s–1), that may occur in stenotic arteries. At shear rates below critical value (1200–1300 s–1), which occur in intact coronary arteries, normally there is no conformational rearrangement of vWF. Pathologic unfolding of vWF at shear rates below critical value may increase a risk of the development of coronary thrombosis. There is little information on the role of shear stress induced conformational rearrangement of vWF in the development of myocardial infarction in young individuals. Purpose To investigate vWF-dependent platelet adhesion of patients with premature myocardial infarction at shear rates below critical value (1200–1300 s–1). Methods Using a microfluidic system, we measured platelet adhesion to a fibrinogen-coated optical surface at shear rates of 1200–1300 s–1 during 10 minutes. We assessed platelet-rich plasma of 8 male persons 40–52 years old, who had previous myocardial infarction at the age of 34–39. The control group comprised 6 healthy male volunteers 30–55 years old. We compared the intensity of scattered laser light measured in volts (V) at 10th minute. To study vWF-dependent platelet adhesion, we blocked GPIb receptor with monoclonal antibody to inhibit platelet interaction with vWF. To compare the intensity of vWF-dependent platelet adhesion with normally occurring adhesion to fibrinogen, we blocked GPIIb/IIIa receptor with monoclonal antibody. Results The inhibition of GPIb vWF-receptor decreased platelet adhesion to fibrinogen surface at shear rates of 1200–1300 s–1 by 17.8±4.7% in healthy volunteers and by 92±2.8% in persons with premature myocardial infarction (p<0.05). Inhibition of GPIIb/IIIa receptor decreased platelet adhesion by 91.5±3.8% in healthy volunteers and by 97.3±3.2% in persons with premature myocardial infarction. Conclusion Pathologic unfolding of vWF at shear rates below critical value may be involved in the development of premature myocardial infarction. Acknowledgement/Funding This work was supported by the grant of the Russian Science Foundation (project #16-15-10098)