Ca2+ sparks promote myogenic tone in retinal arterioles

Background and Purpose

Ca²⁺ imaging reveals subcellular Ca²⁺ sparks and global Ca²⁺ waves/oscillations in vascular smooth muscle. It is well established that Ca²⁺ sparks can relax arteries, but we have previously reported that sparks can summate to generate Ca²⁺ waves/oscillations in unpressurized retinal arterioles, leading to constriction. We have extended these studies to test the functional significance of Ca²⁺ sparks in the generation of myogenic tone in pressurized arterioles.

Experimental Approach

Isolated retinal arterioles (25–40 μm external diameter) were pressurized to 70 mmHg, leading to active constriction. Ca²⁺ signals were imaged from arteriolar smooth muscle in the same vessels using Fluo4 and confocal laser microscopy.

Key Results

Tone development was associated with an increased frequency of Ca²⁺ sparks and oscillations. Vasomotion was observed in 40% of arterioles and was associated with synchronization of Ca²⁺ oscillations, quantifiable as an increased cross-correlation coefficient. Inhibition of Ca²⁺ sparks with ryanodine, tetracaine, cyclopiazonic acid or nimodipine, or following removal of extracellular Ca²⁺, resulted in arteriolar relaxation. Cyclopiazonic acid-induced dilatation was associated with decreased Ca²⁺ sparks and oscillations but with a sustained rise in the mean global cytoplasmic [Ca²⁺] ([Ca²⁺]_c), as measured using Fura2 and microfluorimetry.

Conclusions and Implications

This study provides direct evidence that Ca²⁺ sparks can play an excitatory role in pressurized arterioles, promoting myogenic tone. This contrasts with the generally accepted model in which sparks promote relaxation of vascular smooth muscle. Changes in vessel tone in the presence of cyclopiazonic acid correlated more closely with changes in spark and oscillation frequency than global [Ca²⁺]_c, underlining the importance of frequency-modulated signalling in vascular smooth muscle.     

Site‐specific absence of microfibrillar‐associated protein 4 (MFAP4) from the internal elastic membrane of arterioles in the rheumatoid arthritis synovial membrane: an immunohistochemical study in patients with advanced rheumatoid arthritis versus osteoarthritis

Microfibrillar‐associated protein 4 (MFAP4) is a non‐structural matrix protein with cell regulatory activities and a potential as seromarker for fibrosis. We aimed to study the occurrence of MFAP4 in the synovial membrane from patients with rheumatoid arthritis (RA) vs osteoarthritis (OA). Formaldehyde‐fixed synovial tissue sections, from patients with RA (N = 6) and OA (N = 6) undergoing total hip arthroplasty, were deparaffinized and immunostained with monoclonal antibodies against MFAP4. Elastin was detected using ElastiKit. MFAP4 in serum (sMFAP4) and synovial fluid was measured by an immunoassay. MFAP4 was present in synovial biopsies from both RA and OA patients, particularly prominent in deep arterioles where it colocalized with elastin. Notably however, MFAP4 was absent from the internal elastic lamina in RA arterioles irrespective of disease duration and synovitis activity, while present although with irregular staining patterns in OA specimens. sMFAP4 was increased in RA and OA serum vs healthy controls: median (interquartile range) 29.8 (25.3–39.1) and 25.5 U/L (18.1–43.3) vs 17.7 U/L (13.7–21.2), p = 0.006 and p = 0.02, respectively The concentration of synovial fluid was lower than in serum in both RA and OA. These findings may suggest that MFAP4 is involved in adaptive vessel wall remodeling associated with chronic joint disease.     

介入治疗动脉导管未闭

目的　采用 Amplatzer封堵器和 Porstm ann海绵塞子封堵器方法 ,经皮穿刺动脉、静脉 ,治疗动脉导管未闭(PDA) ,并对其疗效 ,安全性进行评价。方法　 2 0例患者 ,其中男 5例 ,女 15例 ,年龄 10～ 32岁。15例行 Porstm ann法 ,5例做 Am platzer法 ,均经临床症状、体检、EKG(心电图 )、X线胸片及超声心动图检查证实为 PDA。结果　 2 0例患者手术都成功。术后即刻心前区连续性杂音消失。造影示 19例完全堵闭 ,仅 1例存在少量残余分流 ,术后发生溶血 ,经内科保守处理 96小时后超声心动图示分流消失。 2 0例病人随访 6～ 36个月未发现装置移位、再通或狭窄。结论　 Amplatzer封堵器和 Porstmann封堵器经导管介入治疗 PDA是一种安全有效的治疗方法     

Structural changes in renal arterioles in Type I diabetic patients

Aims/hypothesis. We aimed to obtain data on arteriolar structure in a follow-up study of microalbuminuric diabetic patients. Methods. Kidney biopsies were obtained at baseline and after 8 years in 18 Type I (insulin-dependent) diabetic patients. Albumin excretion rate, blood pressure and HbA_1C were measured regularly, and the glomerular filtration rate (GFR) was measured at the time of the kidney biopsy. The biopsy was embedded into plastic blocks and serially sectioned with 1 μm sections. In levels 25 μm apart, afferent and efferent arteriolar profiles were identified and digitised in the electron microscope. The extra-cellular matrix as volume fraction of the media was measured, and estimates of thickness of matrix, media, endothelium and lumen were obtained. Baseline and follow-up biopsies were studied concomitantly. Results. A large increase was seen in matrix volume fraction in afferent (p = 0.0001) and in efferent arterioles (p = 0.0004). Estimated thickness of media and matrix increased, whereas endothelial cell thickness decreased, over the 8 years. There was a correlation between the parameters of diabetic glomerulopathy and arteriolar parameters in the biopsies done at 8 years, basement membrane thickness compared with afferent matrix volume fraction: r = 0.74, p = 0.0005. Also aggravation of glomerulopathy and arteriolar structure over 8 years showed positive correlation. Arteriolar parameters correlated with the albumin excretion rate (AER) and inversely with GFR. Conclusion/interpretation. The arteriolar accumulation of matrix parallels that taking place in glomeruli and shows association with functional parameters over 8 years in Type I diabetic patients with microalbuminuria. These changes are considered an important part of the structural lesions in the diabetic kidney underlying the development of diabetic nephropathy. [Diabetologia (2002) 45: ▪–▪] Received: 9 October 2001 and in revised form: 29 November 2001     

Is Physiological Angiogenesis in Skeletal Muscle Regulated by Changes in Microcirculation?

Physiological angiogenesis occurs in female reproductive organs, in growing antlers as a result of long-term exposure to cold and possibly hypoxia, and due to increased activity (training) in skeletal and cardiac muscle. The common denominator is increased blood flow, which may result in increased velocity of flow and/or diameters in arterioles and capillaries, increased capillary pressure and increased capillary hematocrit. Increased velocity would lead to increased shear stress, while increased pressure and/or diameters would increase wall tension. Either of these factors may cause a disturbance of the endothelium on the luminal side of vessels. In addition, increased contractile activity during training could cause changes on the abluminal side (for example, modification of the capillary basement membrane or the extracellular matrix induced by stretch/relaxation). In order to elucidate the role of these individual factors in angiogenesis, microcirculation was studied in skeletal muscles which were exposed to: (a) increased activity by chronic electrical stimulation; (b) long-term increase in blood flow by various vasodilators; (c) long-term administration of CoCl₂ to increase hematocrit; and (d) long-term stretch, achieved by removal of agonist muscles. Capillary growth, demonstrated as an increased capillary/fiber ratio, as determined by histochemical staining and by electron microscopy, occurred in (a), (b), and (d), but not (c). Capillary proliferation, estimated by labeling index for bromodeoxyuridine of capillary-linked nuclei, occurred in (a), but not in (b). Chronic electrical stimulation resulted in an increase in the diameter of capillaries, a transient widening of arterioles, and no change in venules. Capillary hematocrit and the velocity of red blood cells (V_rbc) were also increased. Calculated shear stress and capillary wall tension were higher in stimulated muscles than in control muscles. Long-term increase in blood flow, induced by administration of the α₁-blocker prazosin, caused increased V_rbc with no change in diameters and increased only capillary shear stress. Stretched muscles had decreased blood flow, but longer sarcomeres initially caused concomitant stretch of capillaries. Increased shear stress/wall tension/stretch may initiate angiogenesis by damaging the luminal side of endothelial cells and/or their basement membrane, or by releasing growth factors or other humoral agents (prostaglandins and/or nitric oxide). Immunohistochemistry in stimulated or stretched muscles showed no evidence for expresson of mRNA for basic fibroblast growth factor (bFGF), or the growth factor itself, but a low molecular mass endothelial cell-stimulating angiogenic factor (ESAF) (77) was increased in (a), (b), and (d). The involvement of prostaglandins and nitric oxide was demonstrated by the finding of attenuated incorporation of BrdU into capillary-linked nuclei in stimulated muscles after administration of indomethacin or L-NNA. Thus, changes in the microcirculation leading to increased shear stress and/or capillary wall tension may stimulate proliferation of endothelial cells either directly, or by release of various humoral factors. However, extravascular mechanical factors have also to be taken into account.     

糖尿病大鼠冠状小动脉超氧阴离子产生量的观察

目的探讨糖尿病大鼠高糖状态下冠状小动脉超氧阴离子(O2-)产生量,阐明高糖状态对O2-产生的影响。方法用链脲佐菌素制备大鼠糖尿病模型,通过手术显微镜及显微手术器械分离大鼠冠状动脉,冷冻切片制备部分标本,用免疫荧光技术测定糖尿病及正常大鼠冠状动脉O2-产生量。结果糖尿病模型组大鼠冠状动脉O2-产生量与正常对照组大鼠及模型组预先加入自由基清除剂组相比明显增加;冷冻切片冠状动脉横切面荧光染色结果表明糖尿病大鼠高糖状态下血管平滑肌与内皮O2-产生均增多。结论高糖能刺激冠状小动脉产生过量的O2-,参与糖尿病状态下的氧化应激,可能与糖尿病微血管并发症密切联系。     

Endothelium-dependent, shear-induced vasodilation is rate-sensitive

OBJECTIVES: To quantify the relative contributions of the rate of change and the magnitude of shear stress to endothelium-mediated arteriolar dilation. METHODS: A feedback control system was designed in which shear stress (tau) and the temporal shear gradient (TSG) were prescribed and dynamically controlled in isolated rat cremaster 1A arterioles. The TSG was the quotient of the maximum shear stress and the ramp duration. This system was used to assess the roles of tau and TSG in the initial, transient vasodilations and the secondary, sustained vasodilations in response to steps and ramps in shear stress. RESULTS: Both step- and ramp-shear experiments revealed time-dependent hiphasic vasodilations that we report for the first time. Application of a step-shear stress of 20 dynes/cm2 elicited an initial transient vasodilation that peaked at about 4 min. When the shear stress was applied as a ramp that reached the maximum value of 20 dynes/cm2 over 5 min, a vasodilation was observed over the ramp period, which reached a peak at the end of the ramp period that was much lower than that observed after step shear. After 20 dynes/cm2 was attained, the vessel diameter decreased despite constant maintenance of the maximum shear stress. In both step- and ramp-shear experiments, after the decrease of the initial vasodilation, a second phase of vasodilation began approximately 15 min after the beginning of the shear application. The second phase of vasodilation reached a steady state that was essentially the same for both the step and the ramp shear. By refining the ramping apparatus further, we varied the TSG up to 40 dynes/cm2 per second and showed that the early vasodilation was highly rate sensitive to TSGs greater than 5 dynes/cm2 per second for a given intermediate value of final shear stress (20 dynes/cm2) and was magnitude sensitive when shear was increased gradually (TSG < 5 dynes/cm2 per second). CONCLUSIONS: Our results suggest that two fundamentally different responses to shear stress are mediated by microvascular endothelium: one vasodilation is elicited by shear stress changes on a time scale of a few seconds or less and another is elicited by shear stress changes on a longer time scale. The former response is potent, transient, and rate sensitive; the latter is more modest, sustained, and magnitude sensitive.