Unveiling the vulnerability of C57BL/6J female mice to HFpEF and its related complications.

Introduction: The impact of female biological sex on the development of heart failure with preserved ejection fraction (HFpEF) and its associated kidney disease and vascular endothelial dysfunction is still controversial. Whether females are protected from HFpEF and associated complications is not well established. Previous studies report conflicting prevalence between genders. We hypothesize that female mice are unprotected from HFpEF and its associated kidney disease and vascular endothelial dysfunction. Methods: Eight-week-old female mice were divided into four groups: control groups receiving a standard diet and water for either 5 or 16 weeks, and HFpEF groups fed a high-fat diet (HFD, Rodent Diet With 60 kcal% Fat) and N [w]-nitro-l-arginine methyl ester (L-NAME - 0.5 g/L) in the drinking water for 5 or 16 weeks. Various measurements and assessments were performed, including echocardiography, metabolic and hypertensive evaluations, markers of heart and kidney injury, and assessment of vascular endothelial function. Results: Female mice with HFD and L-NAME developed HFpEF at 5 weeks, evidenced by increased E/E' ratio, reduced cardiac index, left ventricular mass, and unchanged ejection fraction. After 16 weeks, HFpEF worsened. Metabolic disorders, hypertension, lung wet/kidney weight increase, exercise intolerance, and cardiac/renal injury markers were observed. Vascular endothelial dysfunction was associated with ER stress and fibrosis induction. Conclusions: We found that female mice are susceptible to the development of HFpEF and its associated kidney disease and vascular endothelial dysfunction. Our data support the concept that the female sex does not protect from HFpEF and its associated kidney disease and vascular endothelial dysfunction when disease risk factors are present.

Plasmacytoid dendritic cells contribute to vascular endothelial dysfunction in type 2 diabetes.

Objective: Type 2 diabetes (T2D) is associated with an increased risk of cardiovascular disease due to macro- and microvascular dysfunction. This study aimed to investigate the potential involvement of plasmacytoid dendritic cells (pDCs) in T2D-related vascular dysfunction. Approach and results: pDCs were isolated from db/db and control mice. It was found that pDCs from db/db mice impaired endothelial cell eNOS phosphorylation in response to ATP and decreased vascular endothelium-dependent relaxation compared to pDCs from control mice. Moreover, isolated CD4+ cells from control mice, when stimulated overnight with high glucose and lipids, and isolated pDCs from db/db mice, display elevated levels of ER stress, inflammation, and apoptosis markers. Flow cytometry revealed that pDC frequency was higher in db/db mice than in controls. In vivo, the reduction of pDCs using anti-PDCA-1 antibodies in male and female db/db mice for 4 weeks significantly improved vascular endothelial function and eNOS phosphorylation. Conclusion: pDCs may contribute to vascular dysfunction in T2D by impairing endothelial cell function. Targeting pDCs with anti-PDCA-1 antibodies may represent a promising therapeutic strategy for improving vascular endothelial function in T2D patients. This study provides new insights into the pathogenesis of T2D-related vascular dysfunction and highlights the potential of immunomodulatory therapies for treating this complication. Further studies are warranted to explore the clinical potential of this approach.

Reciprocal regulation of iNOS and PARP-1 during allergen-induced eosinophilia.

Inducible nitric oxide synthase (iNOS) inhibition was recently shown to exert no effect on allergen challenge in human asthma, raising serious concerns about the role of the protein in the disease. The present study investigated the role of iNOS in ovalbumin-induced eosinophilia from the perspective of its relationship with poly(ADP-ribose) polymerase-1 (PARP-1) and oxidative DNA damage. A mouse model of ovalbumin-induced eosinophilia was used to conduct the studies. iNOS-associated protein nitration and tissue damage were partially responsible for allergen-induced eosinophilia. iNOS expression was required for oxidative DNA damage and PARP-1 activation upon allergen challenge. PARP-1 was required for iNOS expression and protein nitration, and this requirement was connected to nuclear factor-kappaB. PARP-1 was an important substrate for iNOS-associated by-products after ovalbumin-challenge. PARP-1 nitration blocked its poly(ADP-ribosyl)ation activity. Interleukin-5 re-establishment in ovalbumin-exposed PARP-1(-/-) mice reversed eosinophilia and partial mucus production without a reversal of iNOS expression, concomitant protein nitration or associated DNA damage. The present results demonstrate a reciprocal relationship between inducible nitric oxide synthase and poly(ADP-ribose) polymerase-1 and suggest that expression of inducible nitric oxide synthase may be dispensable for eosinophilia after interleukin-5 production. Inducible nitric oxide synthase may be required for oxidative DNA damage and full manifestation of mucus production. Such dispensability may explain, in part, the reported ineffectiveness of inducible nitric oxide synthase inhibition in preventing allergen-induced inflammation in humans.

Involvement of Rho-kinase and the actin filament network in angiotensin II-induced contraction and extracellular signal-regulated kinase activity in intact rat mesenteric resistance arteries.

We have previously shown that angiotensin II (Ang II) and pressure increase extracellular signal-regulated kinase (ERK) 1/2 activity synergistically in intact, pressurized resistance arteries in vitro. However, the mechanisms by which pressure and Ang II activate ERK1/2 in intact resistance arteries remain to be determined. The purpose of the present study was to investigate the involvement of Rho-kinase and the actin filament network in Ang II- and pressure-induced ERK1/2 activation, as well as in the contractile response induced by Ang II. Mesenteric resistance arteries (200 to 300 microm) were isolated, mounted in an arteriograph, and stimulated by pressure, Ang II, or both. Activation of ERK1/2 was then measured by an in-gel assay. In mesenteric resistance arteries maintained at 70 mm Hg, Ang II (0.1 micromol/L) induced contraction (29+/-1.4% of phenylephrine, 10 micromol/L-induced contraction) and significantly increased ERK1/2 activity. Selective inhibition of Rho-kinase by Y-27632 (10 micromol/L) and selective disruption of the actin filament network by cytochalasin B (10 micromol/L) both decreased the Ang II-induced contraction by 78+/-1.2% and 87+/-1.9%, respectively, and significantly diminished ERK1/2 activity. In the absence of Ang II, increasing intraluminal pressure from 0 to 70 or 120 mm Hg increased ERK1/2 activity. ERK1/2 activity at 120 mm Hg was similar to that observed at 70 mm Hg in the presence of Ang II. Pressure-induced ERK1/2 activation was markedly attenuated by cytochalasin B but not by Y-27632. These results indicate that whereas pressure-induced ERK1/2 activation requires an intact actin filament network, but not Rho-kinase, the activation of ERK1/2 and the contraction induced by Ang II require both Rho-kinase and an intact actin filament network in isolated, intact mesenteric resistance arteries.

Flow (shear stress)-induced endothelium-dependent dilation is altered in mice lacking the gene encoding for dystrophin.

BACKGROUND: Dystrophin has a key role in striated muscle mechanotransduction of physical forces. Although cytoskeletal elements play a major role in the mechanotransduction of pressure and flow in vascular cells, the role of dystrophin in vascular function has not yet been investigated. Thus, we studied endothelial and muscular responses of arteries isolated from mice lacking dystrophin (mdx mice). METHODS AND RESULTS: Carotid and mesenteric resistance arteries 120 micrometer in diameter were isolated and mounted in vitro in an arteriograph to control intraluminal pressure and flow. Blood pressure was not affected by the absence of dystrophin. Pressure-induced (myogenic), phenylephrine-induced, and KCl-induced forms of tone were unchanged. Flow (shear stress)-induced dilation in arteries isolated from mdx mice was decreased by 50% to 60%, whereas dilation to acetylcholine or sodium nitroprusside was unaffected. NG-nitro-L-arginine methyl ester-sensitive flow dilation was also decreased in arteries from mdx mice. Thus, the absence of dystrophin was associated with a defect in signal transduction of shear stress. Dystrophin was present in vascular endothelial and smooth muscle cells, as shown by immunolocalization, and localized at the level of the plasma membrane, as seen by confocal microscopy of perfused isolated arteries. CONCLUSIONS: -This is the first functional study of arteries lacking the gene for dystrophin. Vascular reactivity was normal, with the exception of flow-induced dilation. Thus, dystrophin could play a specific role in shear-stress mechanotransduction in arterial endothelial cells. Organ damage in such diseases as Duchenne dystrophy might be aggravated by such a defective arterial response to flow.

Axial stretch modifies contractility of porcine coronary arteries by a protein kinase C-dependent mechanism.

Large coronary arteries undergo marked circumferential and axial deformations due to changes in blood pressure and gross movements of the ventricular wall during systole and diastole. The present study was designed to investigate 1) whether axial stretch of large coronary arteries influences the sensitivity to vasoconstrictors, 2) the mechanisms mediating stretch-dependent changes in vascular sensitivity. Endothelium-denuded cylindrical segments from large porcine coronary arteries were studied under isometric conditions using a balloon-based impedance planimetric technique. In segments subjected to a pressure of 60 mmHg, 20% axial stretch caused a left-ward shift of the concentration-response curves for K+ and 5-hydroxytryptamine (5-HT). Enhancement of vascular sensitivity to 5-HT induced by axial stretch was observed also in maximally K+-depolarized coronary arteries. Protein kinase C inhibition by calphostin C (1 microM) slightly decreased the spontaneous resting tone at 60 mmHg and inhibited the leftward shift of the concentration-response curve for 5-HT elicited by axial stretch. These results suggest that axial stretch of the vessel wall enhances the sensitivity of coronary arteries to vasoconstrictors by a protein kinase C-dependent mechanism.

High NaCl intake decreases both flow-induced dilation and pressure-induced myogenic tone in resistance arteries from normotensive rats: involvement of cyclooxygenase-2.

The effect of high NaCl diet on resistance arteries is not yet fully documented. In order to assess the effect of NaCl on myogenic tone and flow-induced dilation independent of arterial blood pressure change, we used normotensive rats which did not develop hypertension upon high NaCl intake. Normotensive Wistar Kyoto Rats received a high (8%) or a normal NaCl diet (0.4%). Mesenteric resistance arteries (150 microm, internal diameter) were cannulated in an arteriograph to allow perfusion of arteries under controlled pressure and flow. Pressure-induced myogenic tone was lower in the high NaCl group than in the control group. Cyclooxygenase inhibition with indomethacin and (N-(2-cyclohexyloxy)-4-nitro-phenyl)-methanesulphonamide, 1 micromol/l) NS 398 (specific cyclooxygenase-2 inhibitor) similarly decreased myogenic tone in rats fed high NaCl but had no effect in those fed a normal NaCl diet. Flow-induced dilation was decreased in the high NaCl group. Inhibition of nitric oxide synthesis with N(G)-nitro-L-arginine methyl ester decreased flow-induced dilation in both groups. Indomethacin and NS 398 did not change flow-induced dilation. As shown by immunofluorescence COX-2 was present in the endothelium of arteries from rats with a high NaCl diet but not in those fed a normal NaCl diet. Thus, chronic high NaCl intake decreased both flow-induced dilation and myogenic tone in resistance arteries. The chronic high NaCl did not affect the participation of nitric oxide on flow-induced dilation, but induced the expression of cyclooxygenase-2, which participates in myogenic tone. These results suggest that high NaCl changes flow and pressure mechanosensing processes and strengthen the hypothesis that sodium ions have an important role in both pressure and flow-mechanotransduction in vascular cells.

Angiotensin II stimulates extracellular signal-regulated kinase activity in intact pressurized rat mesenteric resistance arteries.

The activation of extracellular signal-regulated kinases 1/2 (ERK1/2) was assessed in isolated rat mesenteric resistance arteries (200-micrometer diameter) in a pressure myograph and stimulated for 5 minutes by angiotensin II (Ang II, 0.1 micromol/L) with a pressure of 70 mm Hg. ERK1/2 activity was measured by using an in-gel assay, and ERK1/2 phosphorylation was measured by Western blot analysis with use of a phospho-specific ERK1/2 antibody. Ang II (0.1 micromol/L) induced contraction (28% of phenylephrine contraction, 10 micromol/L). ERK kinase inhibitor PD98059 (10 micromol/L) attenuated this contraction by 36% but not that to phenylephrine or K(+) (60 mmol/L). In unpressurized arteries, Ang II increased ERK1/2 activity by 26%, and pressure (70 mm Hg) itself increased ERK1/2 activity by 72%. Ang II and pressure together acted synergistically, increasing ERK1/2 activity by 264%. Thus, in pressurized vessels, Ang II (0.1 micromol/L) increased ERK1/2 activity by 112%, calculated as [(364/172)-1]x100, which was confirmed by a measured 72% increase in ERK1/2 phosphorylation. Ang II type 1 receptor blockade by candesartan (10 micromol/L) abolished the Ang II-induced increase in ERK1/2 activity, but Ang II type 2 receptor blockade (PD123319, 10 micromol/L) did not. The Ang II-induced increase in ERK1/2 activity was inhibited by protein kinase C inhibitors Ro-31-8220 (1 micromol/L) and Go-6976 (300 nmol/L) and tyrosine kinase inhibitors genistein (1 micromol/L, general) and herbimycin A (1 micromol/L, c-Src family). The present findings show for the first time in intact resistance arteries that ERK1/2 activation is rapidly regulated by Ang II, is synergistic with pressure, and is involved in contraction. The ERK1/2 signaling pathway apparently includes upstream protein kinase C and c-Src.

Tissue angiotensin II and endothelin-1 modulate differently the response to flow in mesenteric resistance arteries of normotensive and spontaneously hypertensive rats.

In resistance arteries pressure-induced (myogenic) tone (MT) and flow (shear stress)-induced dilation (FD) are potent determinant of vascular resistance. We investigated the role of angiotensin II and endothelin-1 in FD and MT in resistance arteries and their potential change in hypertension. Flow - diameter - pressure relationship was established in situ, under anaesthesia, in two daughter branches of a mesenteric resistance artery (180 microM, n=7 per group) from spontaneously hypertensive (SHR) or normotensive (WKY) rats. One artery was ligated distally, so that it was submitted to pressure only, while the other was submitted to pressure and flow. Drugs were added to the preparation and external diameter, pressure and flow measured continuously. External diameter (with flow) ranged from 150+/-3 to 191+/-7 microM in WKY (n=28) rats and from 168+/-6 to 186+/-6 microM in SHR (n=28). Flow induced a dilation of the non-ligated arteries which was lower in SHR (13+/-5 - 31+/-4 microM vs WKY: 5+/-5 - 44+/-4 microM). In the ligated artery, the diameter did not significantly change, due to MT. In the vessels submitted to flow angiotensin converting enzyme inhibition (perindopril, 10 micromol L(-1)) increased the diameter in SHR (+11+/-2 microM) significantly more than in WKY (+2+/-1 microM). Angiotensin type 1 receptor (AT(1)R) blockade (losartan, 10 micromol L(-1)) increased the diameter in the vessels with flow in SHR only (+6+/-1 microM). Angiotensin type 2 receptor (AT(2)R) blockade (PD 123319, 1 micromol L(-1)) decreased arterial diameter in WKY only (9+/-2). Endothelin-1 type A receptor (ET(A)R) blockade (LU135252, 0.1 micromol L(-1)) increased the diameter only in SHR in the artery submitted to flow (by 6+/-1 microM). Thus FD was counteracted by a flow-dependent AT(1) and ET(A) receptors-activation in SHR whereas in WKY FD AT(2)-dependent dilation is involved.

Activation of AT(2) receptors by endogenous angiotensin II is involved in flow-induced dilation in rat resistance arteries.

Pressure-induced tone (myogenic, MT) and flow (shear stress)-induced dilation (FD) are potent modulators of resistance artery tone. We tested the hypothesis that locally produced angiotensin II interacts with MT and FD. Rat mesenteric resistance arteries were perfused in situ. Arterial diameter was measured by intravital microscopy after a bifurcation on 2 distal arterial branches equivalent in size (150 microm, n=7 per group). One was ligated distally and thus submitted to pressure only (MT, no FD). The second branch was submitted to flow and pressure (MT and FD). The difference in diameter between the 2 vessels was considered to be FD. Flow-diameter-pressure relationship was established in the absence and then in the presence of 1 of the following agents. In the nonligated segment (MT+FD), angiotensin II type 1 (AT(1)) receptor blockade (losartan) had no significant effect, whereas angiotensin II type 2 (AT(2)) receptor blockade (PD123319) or saralasin (AT(1)+AT(2) blocker) decreased the diameter significantly, by 9+/-1 and 10+/-0.8 microm, respectively. Angiotensin II in the presence of losartan increased the diameter by 18+/-0.6 microm (inhibited by PD123319). PD123319 or saralasin had no effect after NO synthesis blockade or after endothelial disruption. In the arterial segment ligated distally (MT only), AT(1) or AT(2) receptor blockade had no significant effect. AT(2)-dependent dilation represented 20% to 39% of FD (shear stress from 22 to 37 dyn/cm(2)), and AT(2)-receptor mRNA was found in mesenteric resistance arteries. Thus, resistance arterial tone was modulated in situ by locally produced angiotensin II, which might participate in flow-induced dilation through endothelial AT(2) receptor activation of NO release.

Chronic blockade of endothelin ETA receptors improves flow dependent dilation in resistance arteries of hypertensive rats.

OBJECTIVE: Flow (shear stress)-induced dilation (FD) is attenuated in hypertension. Flow triggers the release by endothelial cells of dilators, such as NO or cyclo-oxygenase (COX) derivatives and constrictor factors such as endothelin-1 (ET-1) which might be involved in several cardiovascular diseases. We hypothesized that ET-1 might play a functional role in FD and participate in the endothelial dysfunction in hypertension. METHODS: We investigated the effect of chronic treatment with the ETA receptor blocker LU135252 (50 mg/kg/day) for 2 weeks on the dilator response to flow in normotensive (Wistar-Kyoto; WKY) or hypertensive (SHR, n = 7 or 8 per group) rats. RESULTS: Systolic arterial pressure was not significantly affected by chronic ETA receptor blockade in both strains. In mesenteric resistance arteries (diameter: approximately 100 microns), isolated in vitro, FD was lower and myogenic tone higher in SHR than in WKY rats. Chronic ETA receptor blockade increased FD by 73% (7.5 +/- 1.5 to 13.0 +/- 2.7 microns dilation with a flow-rate of 150 microliters/min) in SHR (no effect in WKY). The participation of NO to FD was increased in SHR and the participation of dilator COX product(s) (blocked by indomethacin 10 mumol/l) to FD was significantly increased in SHR and in WKY. In control rats FD was improved by acute ETA receptor blockade in WKY rats (18.5 +/- 2.0 to 23.2 +/- 1.8 microns dilation to flow-rate of 150 microliters/min) and significantly more in SHR (6.0 +/- 1.8 to 15.1 +/- 1.6 microns). Acetylcholine-induced dilation was also improved by chronic ETA receptor blockade (no effect of an acute blockade). Myogenic and phenylephrine-induced tone were not affected by chronic or acute ETA receptor blockade. The improvement of endothelium-dependent dilation was not related to a change in blood pressure. CONCLUSION: Chronic ETA receptor blockade increased flow-induced dilation in SHR possibly by suppressing flow-induced ETA stimulation and by improving the release of dilator products by the endothelium.

Indapamide improves flow-induced dilation in hypertensive rats with a high salt intake.

OBJECTIVE: Spontaneously hypertensive rats (SHR) are sensitive to a high salt intake and we investigated the question of whether flow-induced dilation is affected by this type of diet, as flow responses are especially sensitive to small changes in extracellular sodium concentrations. METHODS: We evaluated the effects of a diuretic (indapamide, 1.5 mg/kg per day, 8 weeks) on four groups of SHR (n=42). One group was fed with a normal-salt diet (0.4%, control group, n=10), the second with a high-sodium diet (8%, n=12), the third with a high-sodium diet and indapamide (1.5 mg/kg per day, 8% salt, n=10) and the fourth group was fed with indapamide alone (1.5 mg/kg per day, n=10). The response to flow was studied in mesenteric resistance arteries (146+/-6.1 microm internal diameter, pressure 100 mmHg) cannulated in vitro in an arteriograph. RESULTS: The increase in mean arterial pressure (from 186+/-4 to 218+/-6 mmHg; P < 0.01) and heart weight: body weight ratio (3.48+/-0.09 versus 4.34+/-0.1 mg/g; P< 0.01) caused by the high salt intake was prevented by indapamide. A high salt intake significantly decreased flow-induced dilation (6+/-0.8 versus 10.7+/-1.2 microm dilation with a flow of 160 microl/min; P< 0.05), while indapamide significantly prevented the decrease in flow-induced dilation in high-salt SHR. Indapamide had no significant effect on flow-induced dilation in mesenteric resistance arteries from SHR with a normal-salt diet. CONCLUSIONS: Indapamide prevented the decrease in flow-induced dilation caused by a high-salt diet Therefore, indapamide might counteract the disturbance in sodium-sensitive flow sensor(s), through a diuretic effect.

High sodium intake decreases pressure-induced (myogenic) tone and flow-induced dilation in resistance arteries from hypertensive rats.

High sodium intake has been associated with a higher blood pressure level. Resistance arteries are the main determinants of blood pressure. They are largely regulated by pressure (tensile stress)-induced tone (myogenic tone, MT) and by flow (shear stress)-induced dilation (FD). Thus, we studied the effect of NaCl (8%) intake for 8 weeks on FD and MT in mesenteric resistance arteries of spontaneously hypertensive rats. Arteries were cannulated and mounted in an arteriograph. Intraluminal diameter was measured continuously. High NaCl intake increased mean arterial pressure (186+/-5 to 217+/-6 mm Hg, P<0.01). Passive arterial diameter ranged from 112+/-6 to 185+/-9 microm (pressure from 25 to 125 mmHg, no effect of NaCl). MT developed in response to pressure (tone from 89+/-1% to 83+/-3% of passive diameter, 25 to 125 mm Hg). High NaCl intake significantly decreased MT (89+/-1% versus 83+/-3% of passive diameter when pressure was 125 mm Hg, P<0.023). High NaCl intake also decreased FD (6.5+/-0.8 versus 10+/-1.3 microm dilation under a pressure of 100 mm Hg and a flow rate of 160 microL/min, P<0.012). Thus, high salt intake decreased both flow (shear stress)-induced dilation and pressure (tensile stress)-induced tone in mesenteric resistance arteries. These findings might reflect attenuation by NaCl of flow and pressure mechanosensor processes.

Impaired nitric oxide- and prostaglandin-mediated responses to flow in resistance arteries of hypertensive rats.

In human and experimental hypertension, flow (shear stress)-induced dilation in large arteries is attenuated and resistant to nitric oxide blockade. We tested the hypothesis that a defect in nitric oxide-and/or prostaglandin-dependent flow-induced dilation might occur in mesenteric resistance arteries from spontaneously hypertensive rats (SHR). We measured resistance mesenteric artery diameter in situ by intravital microscopy and simultaneously measured mesenteric arterial pressure in a collateral artery. The flow-diameter-pressure relationship was established in normotensive Wistar-Kyoto rats (WKY) and in SHR under control conditions and after endothelium removal, inhibition of nitric oxide synthesis with N omega-nitro-L-arginine methyl ester (10 micromol/L), or inhibition of prostaglandin synthesis with indomethacin (10 micromol/L). Production of prostaglandins was determined in the perfusate. Endothelium removal decreased artery diameter by 14 +/- 1.6% in WKY and 5 +/- 0.5% (P<.01 versus WKY) in SHR at a flow rate of 400 microL/min. In WKY, N omega-nitro-L-arginine methyl ester and indomethacin decreased resistance artery diameter by 12 +/- 3% (P<.001) and 5 +/- 2% (P<.01), respectively, at a flow rate of 400 microL/min; neither substance had any significant effect in SHR. In both strains, flow induced the production of 6-keto-prostaglandin F1alpha, the metabolite of prostacyclin; prostaglandin F2alpha; and thromboxane B2, the stable metabolite of thromboxane A2. Production of 6-keto-prostaglandin F1alpha and prostaglandin F2alpha was significantly lower in SHR than WKY, and TxB2 production was significantly higher in SHR than WKY. The present findings suggest that in SHR mesenteric resistance arteries, dilation in response to increases in flow was resistant to nitric oxide and prostaglandin synthesis blockade. A modification of the ratio of vasodilator to vasoconstrictor prostaglandins might be at least partly responsible for the decreased dilator response to flow in SHR.

Impaired flow-induced dilation in mesenteric resistance arteries from mice lacking vimentin.

The intermediate filament vimentin might play a key role in vascular resistance to mechanical stress. We investigated the responses to pressure (tensile stress) and flow (shear stress) of mesenteric resistance arteries perfused in vitro from vimentin knockout mice. Arteries were isolated from homozygous (Vim-/-, n = 14) or heterozygous vimentin-null mice (Vim+/-, n = 5) and from wild-type littermates (Vim+/+, n = 9). Passive arterial diameter (175+/-15 micron in Vim+/+ at 100 mmHg) and myogenic tone were not affected by the absence of vimentin. Flow-induced (0-150 microl/min) dilation (e. g., 19+/-3 micron dilation at 150 mmHg in Vim+/+) was significantly attenuated in Vim-/- mice (13+/-2 micron dilation, P < 0.01). Acute blockade of nitric oxide synthesis (NG-nitro- L-arginine, 10 microM) significantly decreased flow-induced dilation in both groups, whereas acute blockade of prostaglandin synthesis (indomethacin, 10 microM) had no significant effect. Mean blood pressure, in vivo mesenteric blood flow and diameter, and mesenteric artery media thickness or media to lumen ratio were not affected by the absence of vimentin. Thus, the absence of vimentin decreased selectively the response of resistance arteries to flow, suggesting a role for vimentin in the mechanotransduction of shear stress.

Rigor tension in single skinned rat cardiac cell: role of myofibrillar creatine kinase.

OBJECTIVE: To elucidate the role of bound creatine kinase in adenine nucleotide compartmentation in myofibrils, the effects of this enzyme's substrates and products on rigor tension were studied in using isolated skinned rat cardiomyocytes rather than fibers, to avoid restrictions due to concentration gradients within the multicellular preparations. METHODS: A new experimental set-up was built to allow continuous and stable measurements of force developed by cells. Triton X-100-treated cardiomyocytes were glued between a glass holder and the needle of a galvanometer. A feedback system allowed the precise measurement of force by recording the coil current necessary to prevent movement of the needle. RESULTS: At very low [Ca2+] (pCa 7), as MgATP level decreased, rigor tension appeared. In the absence of phosphocreatine (PCr), this tension started to rise at MgATP concentrations several times higher than in the presence of 12 mM PCr. In the absence of PCr, the pMgATP/tension curves of single cells usually had a complicated relationship which could not be analyzed by a simple Hill equation. In the absence of PCr, 250 microM MgADP strongly potentiated rigor tension development in the 1 mM-3 microM range of [MgATP]; at 100 microM MgATP, in the presence of MgADP, the tension was 4.6 times higher than in the absence of MgADP. Addition of 12 mM PCr immediately eliminated rigor. Finally, in the presence of 100 microM MgATP and 250 microM MgADP, a decrease in PCr resulted in rigor; the half-maximal contracture being recorded at 1 mM PCr. CONCLUSIONS: These results indicate a myofibrillar compartmentation of adenine nucleotides influenced by bound creatine kinase, since at equal MgATP concentrations in extramyofibrillar milieu the response of myofibrils strongly depends on the presence of PCr. Local accumulation of ADP in myofibrils due to a fall in cellular PCr and inability of myofibrillar creatine kinase to rephosphorylate ADP produced by myosin ATPase could be an important mechanism of diastolic tension rise in ischaemic conditions.

Rapid effect of LPS-activated macrophages on the reactivity of the rat mesenteric artery.

This study was designed to evaluate the effects of lipopolysaccharide (LPS)-activated macrophages in the perfused mesenteric circulation of the rat. The mesenteric network of anesthetized rats was perfused in situ under constant flow conditions and the diameter and pressure of the mesenteric artery were continuously recorded. For the first 30 min the mesenteric network was perfused with an RPMI solution (control condition); thereafter it was perfused for 60 min with the same solution containing either (1) LPS (1 microgram/ml), (2) elicited macrophages (10(6) cells/ml), (3) LPS-activated macrophages or (4) supernatants derived from LPS-activated macrophages (SPN). The changes in arterial diameter induced by topical application of phenylephrine (PE, 10 mumol/l) were measured under control conditions and then 30 and 60 min after the onset of perfusions. The intravascular pressure was similarly increased (51 +/- 6%, p < 0.001) by the perfusion of activated macrophages or elicited macrophages but was not affected by perfusion of LPS or SPN. Despite the same level of transmural mesenteric pressure in rats perfused with activated and elicited macrophages, the mesenteric diameter was significantly larger with activated than with elicited macrophages (p < 0.05). Under control conditions, PE induced a marked decrease in arterial diameter from 495 +/- 15 to 265 +/- 13 microns (p < 0.001). Perfusion of LPS, elicited macrophages or SPN did not modify the vascular reactivity to PE. Perfusion of activated macrophages reduced the PE-induced contraction by 77 +/- 6% (p < 0.001). Perfusion of elicited macrophages with a nitric oxide (NO) donor (SIN-1, 10 mumol/l) reproduced the effect of LPS-activated macrophages while addition of an NO scavenger (oxyhemoglobin, 10 mumol/l) prevented the depression of the vascular reactivity to PE by activated macrophages. Finally, activated macrophages preincubated with an inhibitor of NO synthesis (NG-monomethyl-L-arginine); L-NMMA), and then perfused in RPMI solution without L-NMMA had no effect on the PE reactivity of the mesenteric artery suggesting that NO released by activated macrophages directly and rapidly inhibited the contractility of the mesenteric artery. The results of this study demonstrate the opposing effects of macrophages in the mesenteric circulation to increase microvascular resistance by a rheological effect while decreasing the reactivity of the mesenteric artery as a result of NO released by macrophages.