Rat Cremaster Muscle Research Articles

Effects of cell‐free layer width and its variability on wall shear stress in arterioles

It has been reported that the cell‐free layer width and its interface roughness with erythrocyte core may significantly influence wall shear stress in microcirculatory vessels. However, to date, the quantitative information on how the cell‐free layer width and/or its variability affect the wall shear stress is limited. In this study, temporal variation data of the cell‐free layer width in arterioles (ID 50‐29 μm) in rat cremaster muscles were used to investigate effects of the layer width and its variability on the wall shear stress. The coefficient of variation (CV) of the cell‐free layer width was used to quantify the interfacial variability between the cell‐free layer and erythrocyte core. Velocity gradient in the cell‐free layer was assumed to be linear, and thus the wall shear stress was determined with plasma viscosity and slope of the linear velocity gradient. This study was performed under normal (285.7 ± 37.9 s−1) and reduced (14.4 ± 0.5 s−1) flow conditions with and without erythrocyte aggregation induced by dextran infusion. The level of rat erythrocyte aggregation was adjusted to levels found in normal human blood. As expected, the arteriolar wall shear stress decreased exponentially with mean cell‐free layer width. There was a significant effect (P < 0.05) of erythrocyte aggregation on the variability (CV) of the cell‐free layer width, resulting in a significant increase (P < 0.05) of the wall shear stress.

A novel hydrodynamic method for microvascular flow enhancement

We have shown that drag-reducing polymers (DRP) restore perfusion to a stenotic bed by lowering microvascular resistance. We studied whether resistance-lowering by DRP are due to changes in hydrodynamics or vasodilation. During intravital microscopy of rat cremaster muscle (n=18), DRP infusion increased aortic flow (p<0.002), decreased vascular resistance (p<0.01), increased arteriolar diameter (p=0.023), and increased RBC velocity in the arterioles (p<0.04), venules (p<0.003) and capillaries (p<0.02). To investigate whether DRP lowers resistance without involvement of shear (nitric oxide [NO])-mediated vasodilation, L-NAME was infused in 19 rats, but failed to abolish DRP resistance-lowering. To further investigate whether DRP resistance-lowering depends on vasodilation, adenosine was infused into rabbit femoral arteries (n=19) prior to DRP to achieve marked vasodilation. DRP caused an additional 14% decrease in femoral vascular resistance (p=0.022). DRP enhance microcirculatory perfusion by lowering vascular resistance. This involves not only some degree of shear-induced vasodilation, but also tone-independent resistance lowering mechanisms, suggesting that DRP favorably alter blood flow hydrodynamics. Modulation of blood flow hydrodynamics to enhance perfusion is unique, and may be of therapeutic value for any condition of compromised blood flow.

Fate Of Culture-Expanded Mesenchymal Stem Cells in The Microvasculature

Vascular delivery of mesenchymal stem cells (MSCs) following myocardial infarction is under clinical investigation. Little is known about the microvascular fate of MSCs. We used intravital microscopy of rat cremaster muscle microcirculation to track intraarterially delivered MSCs. Rat MSCs (average diameter, 23 microm) were bolused into the ipsilateral common iliac artery. Interrogation of an arteriole-venule pair revealed that 92+/-7% (n=6) of MSCs arrest and interrupt flow during first pass at the precapillary level, resulting in decreased flow in the feeding arteriole (velocity decreased from 6.3+/-1.0 to 4.6+/-1.3 mm/sec; P<0.001). MSC deformability evaluated using filtration through polycarbonate membranes revealed that the cortical tension of MSCs (0.49+/-0.07 dyne/cm, n=9) was not different from that of circulating mononuclear cells (0.50+/-0.05 dyne/cm, n=7). When intravital microscopy was performed 3 days following injection, the number of MSCs in the cremaster further decreased to 14% of the initial number, because of cell death in situ. In vivo labeling of the basement membrane revealed that at 1 day, the surviving cells were spread out on the luminal side of the microvessel, whereas at 3 days, they integrated in the microvascular wall. Despite their deformability, intraarterially delivered MSCs entrap at the precapillary level because of their large size, with a small proportion of surviving MSCs integrating in a perivascular niche.

Endothelial cell-derived nitric oxide mobilization is attenuated in copper-deficient rats

The attenuation of endothelium-dependent nitric oxide (NO) mediated vasodilation is a consistent finding in both conduit and resistance vessels during dietary copper (Cu) deficiency. Although the effect is well established, evidence for the mechanism remains circumstantial. This study was designed to determine the relative amount of NO produced in and released from the vascular endothelium. Using the fluorescent NO indicator, 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM), we now demonstrate the effect of a Cu-deficient diet on the production of NO from the endothelium of resistance arterioles. In one group of experiments, control and Cu-chelated lung microvascular endothelial cells (ECs) were used to assay NO production and fluorescence was observed by confocal microscopy. Weanling Sprague-Dawley rats were fed purified diets that were either Cu adequate (6.3 micrograms Cu per gram of food) or Cu deficient (0.3 micrograms Cu per gram of food) for 4 weeks. In the second series of experiments, first-order arterioles were microsurgically isolated from the rat cremaster muscle, cannulated, and pressurized with (3[N-morpholino]propanesulfonic acid) physiologic salt solution (MOPS-PSS). DAF-FM (5 micromol.L-1) was added in the lumen of the vessel to measure NO release. Baseline DAF-FM fluorescence was significantly lower in Cu-chelated ECs than in controls. In response to 10-6 mol.L-1 acetylcholine, fluorescent intensity was significantly less in chelated ECs and in the lumen of Cu-deficient arterioles. The results suggest that production and release of NO by the vascular endothelium is inhibited by a restriction of Cu. This inhibition may account for the attenuated vasodilation previously reported in Cu-deficient rats.

Decreased activity of the smooth muscle Na+/Ca2+ exchanger impairs arteriolar myogenic reactivity

Arteriolar myogenic vasoconstriction occurs when stretch or increased membrane tension leads to smooth muscle cell (SMC) depolarization and opening of voltage-gated Ca(2+) channels. While the mechanism underlying the depolarization is uncertain a role for non-selective cation channels has been demonstrated. As such channels may be expected to pass Na(+), we hypothesized that reverse mode Na(+)/Ca(2+) exchange (NCX) may act to remove Na(+) and in addition play a role in myogenic signalling through coupled Ca(2+) entry. Further, reverse (Ca(2+) entry) mode function of the NCX is favoured by the membrane potential found in myogenically active arterioles. All experiments were performed on isolated rat cremaster muscle first order arterioles (passive diameter approximately 150 mum) which were pressurized in the absence of intraluminal flow. Reduction of extracellular Na(+) to promote reverse-mode NCX activity caused significant, concentration-dependent vasoconstriction and increased intracellular Ca(2+). This vasoconstriction was attenuated by the NCX inhibitors KB-R7943 and SEA 04000. Western blotting confirmed the existence of NCX protein while real-time PCR studies demonstrated that the major isoform expressed in the arteriolar wall was NCX1. Oligonucleotide knockdown (24 and 36 h) of NCX inhibited the vasoconstrictor response to reduced extracellular Na(+) while also impairing both steady-state myogenic responses (as shown by pressure-diameter relationships) and acute reactivity to a 50 to 120 mmHg pressure step. The data are consistent with reverse mode activity of the NCX in arterioles and a contribution of this exchanger to myogenic vasoconstriction.

Hydroxyethyl Starch: The Effect of Molecular Weight and Degree of Substitution on Intravascular Retention In Vivo

Hydroxyethyl starch (HES) solution is characterized by its mean molecular weight (MW), concentration, and degree of substitution (DS). This character varies worldwide. After binding fluorescein-isothiocyanate (FITC-HES), we evaluated the retention rate of three types of 6% HES in the A2 and V2 blood vessels of rat cremaster muscles using intravital microscopy in a mild hemorrhage model (10% of total blood volume). After blood withdrawal, we infused three types of FITC-HES: HES-A (MW 150-200 kDa, DS 0.6-0.68), HES-B (MW 175-225 kDa, DS 0.45-0.55), or HES-C (MW 550-850 kDa, DS 0.7-0.8) before determining the FITC-HES retention rate in the intravital microscope. For V2, the FITC-HES retention rates 120 min after the start of the infusion were 27% +/- 7.2% of baseline values (HES-A), 65% +/- 9.1% (HES-B), and 86% +/- 9.6% (HES-C); for A2 they were 27% +/- 6.6%, 73% +/- 10.2%, and 89% +/- 8.7%, respectively. HES-B and HES-C were retained in the vessels longer than HES-A (P = 0.028 for V2, P = 0.038 for A2 between HES-B and HES-A; P = 0.022 for V2, P = 0.037 for A2 between HES-C and HES-A). There was no difference in the rate of disappearance from the vessels between HES-B and HES-C. HES-B and HES-C are equally retained in the blood vessels. Middle-sized HES-B with low DS and middle substitution pattern stayed in the blood vessels as long as the large-sized HES. HES solutions of varying characters should be examined to optimize HES infusion.

The Effect of Hematoma in Muscle Flap Microcirculation

Free radicals were first mentioned by Healey about the hematoma under skin flaps, trying to explain the blood itself has different effect beyond its space-occupying effect or infection. The aim of this study was to determine whether hematoma would have any effect on neutrophil behavior in muscle flap microcirculation. Rat cremaster muscle flap model was used for measuring intravital different parameters. We used the rat cremaster muscle flap model for direct observation and measurement of rolling and adhesion. The rats were randomized into 6 groups. Groups I and IV: only the cremaster muscle was turned to the abdominal wall (n = 4 and n = 4). Groups II and V: saline was injected under the muscle flap (n = 3 and n = 4). Group III and VI: blood was injected under the muscle flap (n = 4 and n = 5). Two sets of microcirculatory measurements were taken in each group. For the comparisons of the groups, the measurements that were taken at the second hour and at the sixth hour were standardized according to the baseline values of the same groups in the same postcapillary venules. Comparisons within the groups were made by Student t test. Between the groups, analysis of variance (ANOVA) independent test was used.Within groups, the number of adherent neutrophils in the second and sixth hour in hematoma groups were found significantly increased according to the baseline measurements, and the number of rolling neutrophils in the second hour in hematoma group was found increased according to the baseline measurements. Between groups, the number of rolling neutrophils in the second hour was found significantly increased between hematoma and sham/hematoma and control groups and the number of adherent neutrophils in the second and sixth hour was found significantly increased between hematoma and sham/hematoma and control groups.

Point:Counterpoint: There is/is not capillary recruitment in active skeletal muscle during exercise

#### Focus. The key issue here is whether resting skeletal muscle is fully perfused. We will make the case that it is not, and thus there exists a reserve of unperfused capillaries (capillary reserve) that are recruited to carry flow not only by muscle contraction, but also by insulin. #### Our

Impact of gradual blood flow increase on ischaemia–reperfusion injury in the rat cremaster microcirculation model

We aimed to evaluate the impact of gradual blood reperfusion on ischaemia-reperfusion injury and to explain the pathophysiology of reperfusion injury in a rat cremaster muscle microcirculation model. Twenty-four Sprague-Dawley rats weighing 150-200 g were evaluated in three groups. Cremaster muscles were prepared for microcirculatory observations. Group I (n=8, control): no ischemia was induced. Group II (n=8, acute reperfusion): microclamps were applied to the right external iliac vessels for 150 min, then venous and arterial clamps were released at once. Group III (n=8, gradual reperfusion): microclamps were applied to the right external iliac vessels for 150 min, and then the first venous clamp was released; the arterial clamp was opened gradually by a specially designed microclamp holder (Sheey ossicle holding clamp). In all groups, following a wait of 150 min blood flow velocity was measured for 15 min and then the animals were reperfused freely for 1h. Next, red blood cell velocity, vessel diameters, functional capillary perfusion and endothelial oedema index were analysed, and rolling, migrating and adhesing leukocytes and lymphocytes were counted. All observations were videotaped for slow-motion replay. Muscle damage was evaluated histologically. In the acute clamp release group, blood velocities increased up to 600% of their pre-ischaemic values during the post-ischaemia-reperfusion period. The numbers of rolling, adhering and transmigrating leukocytes were significantly higher and histological evaluation revealed more tissue damage in the acute reperfusion group. Depending on histological and microcirculatory findings, gradual reperfusion was confirmed to reduce the intensity of reperfusion injury.

Selective resistance to vasoactive effects of insulin in muscle resistance arteries of obese Zucker (fa/fa) rats

Obesity is related to insulin resistance and hypertension, but the underlying mechanisms are unclear. Insulin exerts both vasodilator and vasoconstrictor effects on muscle resistance arteries, which may be differentially impaired in obesity. To investigate whether vasodilator and vasoconstrictor effects of insulin are impaired in muscle resistance arteries of obese rats and the roles of Akt and endothelial NO synthase (eNOS). Effects of insulin were studied in resistance arteries isolated from cremaster muscles of lean and obese Zucker rats. In arteries of lean rats, insulin increased activity of both NO and endothelin (ET-1), resulting in a neutral effect under basal conditions. In arteries of obese rats, insulin induced endothelin-mediated vasoconstriction (-15 +/- 5% at 1 nM, P < 0.05 vs. lean). Insulin induced vasodilatation during endothelin receptor blockade in arteries of lean rats (20 +/- 5% at 1 nM) but not in those of obese rats. Inhibition of NO synthesis increased vascular tone (by 12 +/- 2%) and shifted insulin-mediated vasoreactivity to vasoconstriction (-25 +/- 1% at 1 nM) in lean rats but had no effect in arteries of obese rats, indicating reduced NO activity. Protein analysis of resistance arteries revealed that insulin-mediated activation of Akt was preserved in obese rats, whereas expression of eNOS was markedly decreased. Vasodilator but not vasoconstrictor effects of insulin are impaired in muscle resistance arteries of obese rats, and this selective impairment is associated with decreased protein levels of eNOS. These findings provide a new mechanism linking obesity to insulin resistance and hypertension.

Endothelium‐independent constriction of isolated, pressurized arterioles by Nω‐nitro‐L‐arginine methyl ester (L‐NAME)

Nitric oxide synthase (NOS) inhibitors cause vasoconstriction in pressurized arterioles with myogenic tone. This suggests either tonic production of NO modulates myogenic tone or a direct, NOS-independent effect of the NOS inhibitors. The nature of the contractile effect of the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM) on pressurised arterioles was investigated. Segments of rat cremaster muscle first-order arteriole were cannulated on glass micropipettes and maintained at an intraluminal pressure of 50, 70 or 120 mmHg. L-NAME and the related compound L-NA (100 microM) constricted pressurized vessels with myogenic tone. Removal of the endothelium did not cause constriction or alter myogenic tone, however the constrictor effect of L-NAME persisted. The constrictor effect of L-NAME was abolished by L-arginine (1 mM). Other NO and cGMP pathway inhibitors, including the nNOS inhibitor 7-nitroindazole (100 muM), the NO scavenger carboxy-PTIO (100 microM), the guanylate cyclase inhibitor ODQ (10 microM) and the cGMP inhibitor Rp-8CPT-cGMPS (10 microM) did not cause constriction of the arterioles. L-NAME caused a small (3-4 mV) but not statistically significant depolarization of the arteriolar smooth muscle at both pressures. The constrictor effect was not prevented by the K(+)-channel antagonist tetraethyl ammonium (TEA, 1 mM) or the K(ATP) channel antagonist glibenclamide (1 microM). These observations demonstrate that L-NAME causes an endothelium- and NOS-independent contraction of vascular smooth muscle in isolated skeletal muscle arterioles. It is suggested that the underlying mechanism relates to an arginine binding interaction.

Temporal and spatial variations of cell-free layer width in arterioles

Separation of red blood cells and plasma in microcirculatory vessels produces a cell-free layer at the wall. This layer may be an important determinant of blood viscosity and wall shear stress in arterioles, where most of the hydraulic pressure loss in the circulatory system occurs and flow regulatory mechanisms are prominent. With the use of a newly developed method, the width of the cell-free layer was rapidly and repeatedly determined in arterioles (10- to 50-microm inner diameter) in the rat cremaster muscle at normal arterial pressure. The temporal variation of the cell-free layer width was non-Gaussian, but calculated mean and median values differed by <0.2 microm. The correlation length of the temporal variations downstream (an indication of mixing) was approximately 30 microm and was independent of pseudoshear rate (ratio of mean velocity to vessel diameter) and of vessel diameter. The cell-free layer width was significantly different on opposite sides of the vessel and inversely related. Increasing red blood cell aggregability reduced this inverse relation but had no effect on correlation length. In the diameter range studied, the mean width of the cell-free layer increased from 0.8 to 3.1 microm and temporal variations increased from 30% to 70% of the mean width. Increased aggregability did not alter either relationship. In summary, the cell-free layer width in arterioles is diameter dependent and shows substantial non-Gaussian temporal variations. The temporal variations increase as diameter increases and are inversely related on opposite sides of the vessel.

Effects of fresh versus banked blood transfusions on microcirculatory hemodynamics and tissue oxygenation in the rat cremaster model

Blood transfusion is recommended to prevent the harmful effects of tissue hypoxia. Whether transfusion alleviates hypoxia at the microcirculatory level is unknown. The objective of this study was to determine whether the effects of fresh blood transfusion on microcirculatory hemodynamics after blood loss are preferable to the effects of banked blood transfusion. Intravital microcirculatory hemodynamics was observed in the rat cremaster muscle flap by observers who were not blinded. Lewis rats were divided into 6 groups (8 rats per group): (1) untreated control; (2) blood withdrawal only (1 mL); (3) blood withdrawal and transfusion of rat fresh blood (1 mL); (4) blood withdrawal and transfusion with 1-day rat banked blood (1 mL); (5) blood withdrawal and transfusion with 1-week rat banked blood (1 mL); and (6) blood withdrawal and transfusion with 2-week rat banked blood (1 mL). Red blood cell velocity, blood flow, vessel diameter, functional capillary perfusion, red blood cell deformability, leukocytes (granulocytes and lymphocytes), and tissue oxygenation levels were monitored before transfusion and for 5 hours afterward. Histology was performed to evaluate tissue inflammation (hematoxylin and eosin [H&E] stain and myeloperoxidase [MPO] immunoassay) and hypoxia (Hypoxyprobe assay). Fresh- and banked blood-transfused groups did not differ in vessel diameters or red blood cell velocities. Functional capillary density was greater in the fresh blood group when compared to 1- and 2-week banked blood-transfused groups. The overall number of granulocytes (rolling, sticking, and transmigrated) was greater in all groups after transfusion (P < .001). Rats transfused with banked blood had a greater number of rolling lymphocytes and more inflammation. Tissue oxygenation levels were improved after fresh blood transfusion in comparison to banked blood (9.5 mm Hg vs 8 mm Hg, P = .02). Fresh blood transfusion is more effective in relieving effects of microcirculatory hypoxia. Banked blood, in particular 2-week stored blood, has limited capacity of improving tissue oxygenation.

Issue Highlights

HomeCirculationVol. 115, No. 3Issue Highlights Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIssue Highlights Originally published23 Jan 2007https://doi.org/10.1161/circ.115.3.287Circulation. 2007;115:287EARLY OUTCOMES OF TRICUSPID VALVE REPLACEMENT IN YOUNG CHILDREN, by Bartlett et al.Few data on the outcome of tricuspid valve replacement in young children are available to guide decisions. Bartlett and colleagues reviewed the results of tricuspid valve replacement in 97 children younger than age 6 years using the database of the Pediatric Cardiac Care Consortium of 45 centers between 1984 and 2002. The primary outcome was survival to discharge. The most frequent cardiac diagnoses were Ebstein’s anomaly (40%), pulmonary atresia (11%), and tetralogy of Fallot (8%). In-hospital mortality was 26% overall and was highest among infants (64%). Valve thrombosis and heart block requiring a pacemaker were common complications. The high morbidity and mortality of tricuspid valve replacement in young children and especially infants suggests that other surgical options should be considered. See p 319.MYOCARDIAL ISCHEMIC MEMORY IMAGING WITH MOLECULAR ECHOCARDIOGRAPHY, by Villanueva et al.Myocardial ischemia that does not result in necrosis may be difficult to diagnose after resolution of the ischemia. Such episodes are associated with endothelial upregulation of leukocyte adhesion molecules. In this issue, Villanueva et al describe the potential of myocardial ischemic memory using contrast echocardiography. Lipid microbubbles were designed to adhere to selectins, and this was tested in a model of inflamed rat cremaster muscle and in a rat model. Twelve rats underwent 15 minutes of left anterior descending coronary artery occlusion and then at 15 minutes and 1 hour following reperfusion, there was greater opacification of the ischemic bed using microbubbles with the selectin ligand. Immunostaining confirmed endothelial P-selectin expression. Although preliminary, the present study raises the potential that this technique may allow the identification of recent myocardial ischemia in patients and facilitate the triage of patients with chest pain. See p 345.PREVALENCE OF LONG-QT SYNDROME GENE VARIANTS IN SUDDEN INFANT DEATH SYNDROME, by Arnestad et al.andCARDIAC SODIUM CHANNEL DYSFUNCTION IN SUDDEN INFANT DEATH SYNDROME, by Wang et al.Sudden infant death syndrome is unexplained, unexpected death in the first year of life and has multifactorial etiologies. One important cause relates to the genes responsible for congenital long-QT syndrome. In companion manuscripts in this issue of Circulation, Arnestad and colleagues report their molecular autopsy findings of genetic screening for 7 known long-QT syndrome—associated genes in 201 Norwegian victims of sudden infant death syndrome. They identified mutations or rare genetic variants in 10% of cases, of which half were in the sodium channel SCN5A gene. Wang and colleagues then performed biophysical in vitro functional experiments on the SCN5A mutations in transiently transfected cells. They found defects in the voltage dependence of inactivation and kinetics, including increased persistent sodium currents, similar to the biophysical changes seen in SCN5A mutations causing long-QT syndrome. Taken together, these investigations conclude that neonatal electrocardiographic screening may identify QT prolongation prior to sudden infant death syndrome, potentially affecting outcome. These studies further our understanding of the relationship between sudden infant death syndrome and genes causing long-QT syndrome. See pp 361 and 368.Visit http://circ.ahajournals.org:Clinician UpdateMetabolic Syndrome. See p e32.Images in Cardiovascular MedicineDouble-Outlet Left Ventricle. See p e36.Serial Images Demonstrating Proximal Extension of an Aortic Intramural Hematoma. See p e38. Download figureDownload PowerPointLost P’s, but Not Yet Forgotten. See p e41.Book ReviewPediatric Prevention of Atherosclerotic Cardiovascular Disease. See p e43.CorrespondenceSee p e45. Previous Back to top Next FiguresReferencesRelatedDetails January 23, 2007Vol 115, Issue 3 Advertisement Article InformationMetrics https://doi.org/10.1161/circ.115.3.287 Originally publishedJanuary 23, 2007 PDF download Advertisement

Preservation of Rat Cremaster Muscle Microcirculation After Prolonged Cold Storage and Transplantation

Preservation of Rat Cremaster Muscle Microcirculation After Prolonged Cold Storage and Transplantation

Properties of the large conductance K+ channel (BKCa) in skeletal muscle arterioles

BKCa channels provide a mechanism for hyperpolarization and relaxation in arteriolar smooth muscle. Thus, BKCa is modulated by Ca2+ sparks providing a negative feedback on myogenic constriction and is a target for endothelium-dependent hyperpolarization. However, discordant results in differing vascular beds and between preparations suggest that heterogeneity may exist in these mechanisms. The present study examined properties of BKCa in rat cremaster muscle arterioles using in vivo and in vitro approaches. Under both conditions the BKCa inhibitor, iberiotoxin (IBTX, 10–7M), caused constriction of the 1st order arteriole. The voltage-gated K+ channel blocker, 4-aminopyridine similarly caused constriction while in vitro studies showed the extent of IBTX and 4-AP-induced constriction to be unaffected by level of intraluminal pressure. Whole cell patch clamp studies on dispersed 1A smooth muscle cells showed that total K+ current was inhibited by <10% by IBTX but blocked approx. 65% by 4-AP. Real-time PCR indicated a lower ratio of beta:alpha subunits compared to that seen in branches of the middle cerebral artery taken from the same animals. Collectively, the data are consistent with a background level of BKCa activity in skeletal muscle arterioles. Further, differences in relative expression of channel subunits between vascular beds suggests that regulation may also occur at the level of transcription.

Effects of supplementation of BH4 after prolonged ischemia in skeletal muscle

To determine whether the supplementation of tetrahydrobiopterin (BH(4), an essential cofactor of nitric oxide synthase; NOS) could attenuate endothelial dysfunction and improve NOS activity and cell viability in skeletal muscle after ischemia/reperfusion (I/R). A vascular pedicle isolated rat cremaster muscle model was used. Cremaster muscles were subjected to 4 h of ischemia followed by 2 h of reperfusion. Rats were given either normal saline or BH(4) by intravenous injection at 1 min prior to reperfusion. After reperfusion, average arteriole diameter, capillary perfusion, endothelial-dependent/-independent vasodilatation, NOS activity, and muscle cell viability were evaluated. Supplementation of BH(4) prior to reperfusion significantly attenuated reperfusion-induced vasoconstriction, poor capillary perfusion, and endothelial dysfunction and enhanced cNOS activity and slightly improved cell viability in the skeletal muscle after I/R. Supplementation of BH(4) during reperfusion provided a significant protection against I/R injury in rat skeletal muscle.

YC-1 Attenuated Post-Ischemic Induced Leukocyte/Endothelial Cell Adhesive Interactions in Rat Cremaster Muscle: Role of GC/cGMP/PKG Signaling Pathway

Ischemia-reperfusion (I/R) injury is a complex process involving the generation and release of inflammatory cytokines, accumulation and infiltration of neutrophils and macrophages, that disturb the microcirculatory hemodynamics. It has been reported that NO-donor induced elevation of intracellular cGMP alleviates I/R injury. An activator of soluble guanylyl cyclase (sGC), YC-1 [3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole] could potentially increase intracellular cGMP levels and thus protect the tissue against I/R injury. Therefore this study aimed to assess the effects of YC-1 on I/R-injured cremaster muscles and further elucidate its underlying mechanisms.

YC-1 Ameliorated I/R-Induced Microcirculatory Disturbance in Rat Cremaster Muscle: A Mechanistic Study

The authors' previous study speculated that YC-1, a guanylate cyclase (GC) activator, ameliorated ischemia-reperfusion (I/R)-induced microcirculatory disturbance; however, its underlying mechanism remains largely unknown. This study aimed to investigate the role of the GC, protein kinase C (PKC), and MAP kinase in YC-1's ability to ameliorate I/R-induced microcirculatory disturbances in rat cremaster muscle flaps.

Integrin Ligands Mobilize Ca2+ from Ryanodine Receptor-gated Stores and Lysosome-related Acidic Organelles in Pulmonary Arterial Smooth Muscle Cells

Extracellular matrix (ECM) protein receptors, or integrins, participate in vascular remodeling and the systemic myogenic response. Synthetic ligands and ECM fragments regulate the vascular smooth muscle cell contractile state by altering intracellular Ca2+ levels ([Ca2+]i). Information on the Ca2+ effect of integrins in vascular smooth muscle cells is limited, but nonexistent in pulmonary arterial smooth muscle cells (PASMCs). We therefore characterized integrin expression in endothelium-denuded pulmonary arteries, and explored [Ca2+]i mobilization pathways induced by soluble ligands in rat PASMCs. Reverse transcriptase-PCR showed mRNA expression of integrins alpha1, alpha2, alpha3, alpha4, alpha5, alpha7, alpha8, alpha(v), beta1, beta3, and beta4, and immunoblots of alpha5, alpha(v), beta1, and beta3 confirmed protein expression. Exposure of PASMCs to integrin-binding peptides (0.5 mM) containing the arginine-glycine-aspartate (RGD) motif elicited [Ca2+]i responses with an order of potency of GRGDNP > GRGDSP > GRGDTP = cyclo-RGD. Pharmacological analysis revealed that the GRGDSP-induced Ca2+ response was unrelated to Ca2+ influx and the inositol triphosphate receptor-gated Ca2+ store, but partially blocked by ryanodine or inhibition of lysosome-related acidic organelles with bafilomycin A1. Simultaneous inhibition of both pathways was necessary to abolish the response. GRGDSP treatment increased cyclic ADP-ribose, the endogenous activator of ryanodine receptors, by 70%. GRGDSP also rapidly reduced Lysotracker Red accumulation, confirming direct modulation of acidic organelles. These data are the first demonstration of integrin-mediated Ca2+ regulation in PASMCs. The presence of an array of integrins, and activation of ryanodine-sensitive Ca2+ stores and lysosome-like organelles by GRGDSP suggest important roles for integrin-dependent Ca2+ signaling in regulating PASMC function.