Baseline Capillary Density Research Articles

Effects of cardiac rehabilitation on in vivo nailfold microcirculation in patients with cardiovascular disease.

This study aimed to explore the impact of cardiac rehabilitation (CR) on in vivo and ex vivo microcirculation, exercise capacity, and oxidative stress in patients with cardiovascular disease (CVD). The study included patients with acute coronary syndrome (ACS; n = 45; age, 69.0 ± 14.1 years) and heart failure (HF; n = 66; age, 77.3 ± 10.7 years) who underwent supervised CR during hospitalization. The control group comprised patients without CVD (NCVD; n = 20; age, 75.9 ± 11.2 years). In vivo microcirculatory observations using nailfold video capillary endoscopy at rest and during hyperemia, exercise capacity, and oxidative stress were assessed at baseline and 12 weeks after discharge. Baseline capillary densities were significantly lower in the ACS (5.0 ± 1.7 capillaries/mm2) and HF (4.9 ± 1.7 capillaries/mm2) groups than in the NCVD group (6.5 ± 1.1 capillaries/mm2, p < 0.01). Similarly, capillary density during reactive hyperemia was significantly lower in the ACS (5.8 ± 1.7 capillaries/mm2) and HF (5.4 ± 1.8 capillaries/mm2) groups than in the NCVD group (7.3 ± 1.4 capillaries/mm2, p < 0.01). Patients with ACS and HF had increased capillary densities at 12 weeks compared with at baseline (p < 0.05). This improvement was particularly pronounced among post-discharge outpatient CR participants (n = 20). Grip strength, exercise capacity, and oxidative stress improved at 12 weeks. Baseline capillary density changes were positively correlated with grip strength changes (r = 0.45, p < 0.001). CR significantly improved nailfold capillary density in patients with ACS and HF 12 weeks after discharge.

NAIL CAPILLARY DENSITY DURING POSTOCCLUSIVE REACTIVE HYPEREMIA AND VENOUS CONGESTION IS MORE IMPAIRED IN DIABETIC COMPARED TO NON-DIABETIC CKD PATIENTS

Objective: Alterations in endothelial function and capillary circulation have been associated with increased cardiovascular events and overall mortality. Both diabetes mellitus (DM) and chronic kidney disease (CKD) have been associated with microcirculatory damage. Nailfold video-capillaroscory can provide a thorough assessment of capillary density and microcirculation changes. This is the first study examining in comparison microcirculatory function parameters in diabetic and non-diabetic patients with CKD. Design and method: We included 48 diabetic and 48 non-diabetic adult patients (&gt;18 years) with CKD (eGFR: &lt; 90 and &gt; 15 mL/min/1.73m2), matched in a 1:1 ratio for age, sex and eGFR within each CKD stage (2, 3a, 3b and 4). All participants underwent nailfold video-capillaroscopy, during which capillary density was measured at normal conditions (baseline), after a 4-minute arterial occlusion (postocclusive reactive hyperemia) and at the end of a 2-minute venous occlusion (congestion phase). Results: Baseline demographic, anthropometric and laboratory characteristics were similar between patients with and without diabetes in total and in CKD stages. Overall, no significant differences at baseline capillary density were observed between groups; however diabetic patients presented significantly lower capillary density during reactive hyperemia (36.3 ± 3.8 vs 38.3 ± 4.3 capillaries/mm2, p = 0.022) and at venous congestion (37.8 ± 4.0 vs 39.8 ± 4.2 capillaries/mm2, p = 0.015). When stratified according to CKD stages, the between-group differences in parameters of interest were not significant in stages 2, 3a and 4. In stage 3b, capillary density was significantly lower in diabetic compared to non-diabetic subjects at baseline (31.1 ± 2.8 vs 33.4 ± 3.4 capillaries/mm2, p = 0.044), during postocclusive hyperemia (36.8 ± 2.7 vs 40.0 ± 4.3 capillaries/mm2, p = 0.037) and venous congestion (38.3 ± 2.8 vs 41.5 ± 3.5 capillaries/mm2, p = 0.022). Conclusions: Capillary density during postocclusive reactive hyperemia and after venous congestion is lower in diabetic compared to non-diabetic CKD patients, a finding indicative that diabetes is an additional factor contributing to microcirculatory functional impairment in CKD. These differences are more prominent in CKD stage 3b, and less prominent in earlier and later stages.

Microcirculatory function deteriorates with advancing stages of chronic kidney disease independently of arterial stiffness and atherosclerosis.

Cardiovascular disease is the main cause of mortality in chronic kidney disease (CKD). Endothelial dysfunction and capillary rarefaction are established cardiovascular risk factors. Nailfold video capillaroscopy provides a thorough assessment of capillary density and functional reserve. This study aimed to examine possible differences in structural and functional capillary density in CKD stages 2-4 with nailfold video capillaroscopy. Ninety-six CKD patients, divided into four equally sized groups according to CKD stage (2, 3a, 3b, 4), underwent nailfold video capillaroscopy, during which capillary density was measured at baseline, after 4-min arterial occlusion and after 2-min venous occlusion. Arterial stiffness and wave parameters were measured with applanation tonometry and commoncarotid intima-media thickness (ccIMT) with ultrasound. Baseline capillary density showed a progressive reduction with advancing CKD stages (stage 2: 32.6 ± 2.8, stage 3a: 31.2 ± 3.8, stage 3b: 32.5 ± 3.3, stage 4: 28.5 ± 3.1, p = 0.011). Similar reductions were observed during postocclusive hyperemia (39.4 ± 3.0, 37.6 ± 4.2, 38.4 ± 3.8, and 33.8 ± 3.3, respectively; p = 0.021) and after venous congestion (41.1 ± 3.1, 39.0 ± 4.4, 39.9 ± 3.5, and 35.2 ± 3.4; p = 0.032). Office PWV and ccIMT showed nonsignificant increasing trends with advancing CKD. In multivariate analysis, eGFR showed a positive association (per ml/min increase; β: 0.053, 95% CI: 0.004-0.101), whereas diabetes (β: -1.706, 95% CI: -3.176 to -0.236) and parathyroid hormone (PTH) (per pg/ml increase; β: -0.022, 95% CI: -0.036 to -0.008) had negative associations with postocclusive capillary density. Both structural and functional capillary density progressively decrease with advancing CKD stages. Apart from reduced eGFR, diabetes and increased PTH levels are independently associated with this reduction. This capillary rarefaction may largely contribute to the increased cardiovascular risk of CKD patients.

P1012NAIL CAPILLARY DENSITY DURING POSTOCCLUSIVE REACTIVE HYPEREMIA AND VENOUS CONGESTION IS MORE IMPAIRED IN DIABETIC COMPARED TO NON-DIABETIC CKD PATIENTS

Abstract Background and Aims Αlterations in endothelial function and capillary circulation have been associated with increased cardiovascular events and overall mortality. Both diabetes mellitus (DM) and chronic kidney disease (CKD) have been associated with microcirculatory damage. Nailfold video-capillaroscory can provide a thorough assessment of capillary density and microcirculation changes. This is the first study examining in comparison microcirculatory function parameters in diabetic and non-diabetic patients with CKD. Method We included 48 diabetic and 48 non-diabetic adult patients (&amp;gt;18 years) with CKD (eGFR: &amp;lt;90 and ≥15mL/min/1.73m2), matched in a 1:1 ratio for age, sex and eGFR within each CKD stage (2, 3a, 3b and 4). All participants underwent nailfold video-capillaroscopy, during which capillary density was measured at normal conditions (baseline), after a 4-minute arterial occlusion (postocclusive reactive hyperemia) and at the end of a 2-minute venous occlusion (congestion phase). Results Baseline demographic, anthropometric and laboratory characteristics were similar between patients with and without diabetes in total and in CKD stages. Overall, no significant differences at baseline capillary density were observed between groups; however diabetic patients presented significantly lower capillary density during reactive hyperemia (36.3±3.8 vs 38.3±4.3 capillaries/mm2, p=0.022) and at venous congestion (37.8±4.0 vs 39.8±4.2 capillaries/mm2, p=0.015). When stratified according to CKD stages, the between-group differences in parameters of interest were not significant in stages 2, 3a and 4. In stage 3b, capillary density was significantly lower in diabetic compared to non-diabetic subjects at baseline (31.1±2.8 vs 33.4±3.4 capillaries/mm2, p=0.044), during postocclusive hyperemia (36.8±2.7 vs 40.0±4.3 capillaries/mm2, p=0.037) and venous congestion (38.3±2.8 vs 41.5±3.5 capillaries/mm2, p=0.022). Conclusion Capillary density during postocclusive reactive hyperemia and after venous congestion is lower in diabetic compared to non-diabetic CKD patients, a finding indicative that diabetes is an additional factor contributing to microcirculatory functional impairment in CKD. These differences are more prominent in CKD stage 3b, and less prominent in earlier and later stages.

Muscle-derived SDF-1α/CXCL12 modulates endothelial cell proliferation but not exercise training-induced angiogenesis.

Chemokines are critical mediators of angiogenesis in several physiological and pathological conditions; however, a potential role for muscle-derived chemokines in exercise-stimulated angiogenesis in skeletal muscle remains poorly understood. Here, we postulated that the chemokine stromal cell-derived factor-1 (SDF-1α/C-X-C motif chemokine ligand 12: CXCL12), shown to promote neovascularization in several organs, contributes to angiogenesis in skeletal muscle. We found that CXCL12 is abundantly expressed in capillary-rich oxidative soleus and exercise-trained plantaris muscles. CXCL12 mRNA and protein were also abundantly expressed in muscle-specific peroxisome proliferator-activated receptor γ coactivator 1α transgenic mice, which have a high proportion of oxidative muscle fibers and capillaries when compared with wild-type littermates. We then generated CXCL12 muscle-specific knockout mice but observed normal baseline capillary density and normal angiogenesis in these mice when they were exercise trained. To get further insight into a potential CXCL12 role in a myofiber-endothelial cell crosstalk, we first mechanically stretched C2C12 myotubes, a model known to induce stretch-related chemokine release, and observed increased CXCL12 mRNA and protein. Human umbilical vein endothelial cells (HUVECs) exposed to conditioned medium from cyclically stretched C2C12 myotubes displayed increased proliferation, which was dependent on CXCL12-mediated signaling through the CXCR4 receptor. However, HUVEC migration and tube formation were unaltered under these conditions. Collectively, our findings indicate that increased muscle contractile activity enhances CXCL12 production and release from muscle, potentially contributing to endothelial cell proliferation. However, redundant signals from other angiogenic factors are likely sufficient to sustain normal endothelial cell migration and tube formation activity, thereby preserving baseline capillary density and exercise training-mediated angiogenesis in muscles lacking CXCL12.

Muscle‐derived SDF‐1α/CXCL12 modulates endothelial cell proliferation but is not required for exercise training‐induced angiogenesis

Chronic elevations in contractile activity, as observed with exercise training, increase capillarity density in skeletal muscle. Here, we demonstrate that the stromal cell‐derived factor‐1 alpha (SDF‐1α/CXCL12) is the most abundantly expressed angiogenesis‐associated chemokine in capillary‐rich oxidative soleus and exercise‐trained plantaris muscles. CXCL12 is also increased in muscle‐specific peroxisome proliferator‐activated receptor γ coactivator 1α transgenic mice (PGC‐1α mTg) when compared to wild‐type littermates. Additionally, CXCL12 is increased in C2C12 myotubes by cyclic mechanical stretch. Furthermore, exposure of human umbilical vein endothelial cells (HUVEC) to conditioned medium from cyclically stretched C2C12 myotubes enhanced cell proliferation. Such an effect was specific to CXCL12 signaling because co‐treatment with AMD3100, an antagonist of the CXCL12 receptor CXCR4, disrupted the proliferative response. To gain further insight into a potential role for CXCL12 in modulating chronic contractile activity‐induced angiogenesis in vivo, we studied muscle‐specific CXCL12 knockout mice (CXCL12 mKO). Interestingly, our results demonstrate that these mice have normal baseline capillary density and preserved angiogenesis when exercise‐trained. These findings are consistent with a muscle‐derived CXCL12‐dependent modulatory role of endothelial cell proliferation in response to increased contractile activity. However, muscle‐derived CXCL12 is not required for exercise training‐induced angiogenesis in skeletal muscle.Support or Funding InformationThis study was supported by Grant‐in‐Aid for Challenging Exploratory Research (16K13019) and Grant‐in‐Aid for Scientific Research (B) (15H03080) (18H03153) to Mitsuharu Okutsu.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Recruitment of non-perfused sublingual capillaries increases microcirculatory oxygen extraction capacity throughout ascent to 7126m.

A physiological response to increase microcirculatory oxygen extraction capacity at high altitude is to recruit capillaries. In the present study, we report that high altitude-induced sublingual capillary recruitment is an intrinsic mechanism of the sublingual microcirculation that is independent of changes in cardiac output, arterial blood pressure or systemic vascular hindrance. Using a topical nitroglycerin challenge to the sublingual microcirculation, we show that high altitude-related capillary recruitment is a functional response of the sublingual microcirculation as opposed to an anatomical response associated with angiogenesis. The concurrent presence of a low capillary density and high microvascular reactivity to topical nitroglycerin at sea level was found to be associated with a failure to reach the summit, whereas the presence of a high baseline capillary density with the ability to further increase maximum recruitable capillary density upon ascent to an extreme altitude was associated with summit success. A high altitude (HA) stay is associated with an increase in sublingual capillary total vessel density (TVD), suggesting microvascular recruitment. We hypothesized that microvascular recruitment occurs independent of cardiac output changes, that it relies on haemodynamic changes within the microcirculation as opposed to structural changes and that microcirculatory function is related to individual performance at HA. In 41 healthy subjects, sublingual handheld vital microscopy and echocardiography were performed at sea level (SL), as well as at 6022m (C2) and 7042m (C3), during ascent to 7126m within 21days. Sublingual topical nitroglycerin was applied to measure microvascular reactivity and maximum recruitable TVD (TVDNG ). HA exposure decreased resting cardiac output, whereas TVD (mean±SD) increased from 18.81±3.92 to 20.92±3.66 and 21.25±2.27mmmm-2 (P<0.01). The difference between TVD and TVDNG was 2.28±4.59mmmm-2 at SL (P<0.01) but remained undetectable at HA. Maximal TVDNG was observed at C3. Those who reached the summit (n=15) demonstrated higher TVD at SL (P<0.01), comparable to TVDNG in non-summiters (n=21) at SL and in both groups at C2. Recruitment of sublingual capillary TVD to increase microcirculatory oxygen extraction capacity at HA was found to be an intrinsic mechanism of the microcirculation independent of cardiac output changes. Microvascular reactivity to topical nitroglycerin demonstrated that HA-related capillary recruitment is a functional response as opposed to a structural change. The performance of the vascular microcirculation needed to reach the summit was found to be associated with a higher TVD at SL and the ability to further increase TVDNG upon ascent to extreme altitude.

Uric acid and skin microvascular function: the Maastricht study.

Microvascular dysfunction has been suggested as a possible underlying mechanism for the association between uric acid and various diseases, such as hypertension, renal disease and cardiomyopathies. We therefore analysed the association between serum uric acid and skin microvascular function, a model of generalized microvascular function. A cross-sectional study was performed in 610 individuals [51.8% men; mean age 58.7 ± 8.6 years; 23.6% with type 2 diabetes (by design)] from The Maastricht Study. We assessed skin capillary density (capillaries/mm) by capillaroscopy at baseline, after 4 min of arterial occlusion, and after 2 min of venous congestion. Capillary recruitment after arterial occlusion and during venous congestion was expressed as the absolute change in capillary density after recruitment and as the percentage change in capillary density from baseline. Crude linear regression analyses showed that serum uric acid [per +1 standard deviation (SD) of 74 μmol/l] was not associated with baseline capillary density [β = -0.21 (95% confidence interval, 95% CI -1.61 to 1.19) P = 0.765], while an inverse association was found between uric acid and absolute change in capillary density after arterial occlusion [β = -1.15 (95% CI -2.36 to 0.06) P = 0.062] and during venous congestion [β = -1.41 (95% CI -2.68 to -0.14) P = 0.029]. However, after adjustment for sex, age and glucose metabolism status, these associations were no longer statistically significant. In addition, we found no association between uric acid and percentage capillary recruitment after arterial occlusion [β = -1.66 (95% CI -3.97 to 0.65) P = 0.159] or during venous congestion [β = -2.02 (95% CI -4.46 to 0.42) P = 0.104] in unadjusted analyses; multivariable analyses gave similar results. These results do not support the hypothesis that generalized microvascular dysfunction (as estimated in skin microcirculation) is the underlying mechanism for the association between uric acid and cardiovascular and renal diseases. The possibility that uric acid is associated with microvascular dysfunction in specific end-organs, for example heart or kidney, needs further investigation.

Proliferative Retinopathy in Type 1 Diabetes Is Associated With Cerebral Microbleeds, Which Is Part of Generalized Microangiopathy

OBJECTIVE We investigated whether proliferative diabetic retinopathy in type 1 diabetic patients can be generalized to cerebral small vessel disease and whether it is associated with impaired peripheral microvascular function. RESEARCH DESIGN AND METHODS Thirty-three patients with proliferative diabetic retinopathy (PDR+), 34 patients without proliferative diabetic retinopathy, and 33 controls underwent magnetic resonance imaging to assess cerebral microangiopathy (cerebral microbleeds) and ischemic damage (white matter hyperintensities and lacunes). Peripheral microvascular function, i.e., skin capillary density and capillary recruitment, was assessed by capillary microscopy. RESULTS Cerebral microbleeds, but not ischemic damage, were more prevalent in PDR+ patients versus the other groups (P < 0.05). A trend was found across groups for the lowest baseline capillary density in PDR+ patients (P for trend = 0.05). In individuals with microbleeds, capillary recruitment was impaired compared with those without microbleeds (P = 0.04). CONCLUSIONS In PDR+ patients, cerebral microbleed prevalence was higher and seems part of generalized microangiopathy that may affect the skin and the brain.

Homocysteine levels are inversely associated with capillary density in men, not in premenopausal women

Homocysteine is an independent predictor of cardiovascular risk. The mechanisms underlying this link are not fully elucidated. Whereas the role of vascular dysfunction in conduit arteries is extensively studied, the role of the microcirculation in this relationship is largely unexplored. We assessed the relationship between homocysteine levels and microvascular structure and function in a healthy, population-based cohort. We cross-sectionally studied 260 participants (aged 42 years, 47% men) of the Amsterdam Growth and Health Longitudinal Study. Nailfold videocapillaroscopy was used to assess capillary density at baseline, during venous occlusion and during peak reactive hyperaemia. The relationship between tertiles of homocysteine and microvascular outcomes was evaluated using linear regression analyses, with adjustment for BMI and blood pressure. Stratified analyses were performed for men and women. In men, we observed a negative, nonlinear relationship between homocysteine and baseline capillary density, showing a lower capillary density in the highest tertile of homocysteine [adjusted B -8.65 capillaries/mm(2) (95%-CI: -16.05 to -1.25); P = 0.02]. In women, no significant associations were found between homocysteine and microvascular outcomes. In men, higher homocysteine levels are associated with a reduction in basal perfusion of skin capillaries. This finding provides a novel potential explanation for how homocysteine influences cardiovascular disease risk.

Id Proteins Regulate Capillary Repair and Perivascular Cell Proliferation following Ischemia-Reperfusion Injury

Acute kidney injury (AKI) results in microvascular damage that if not normally repaired, may lead to fibrosis. The Id1 and 3 proteins have a critical role in promoting angiogenesis during development, tumor growth and wound repair by functioning as dominant negative regulators of bHLH transcription factors. The goal of this study was to determine if Id proteins regulate microvascular repair and remodeling and if increased Id1 expression results in decreased capillary loss following AKI. The effect of changes in Id expression in vivo was examined using Id1−/−, Id3RFP/+ (Id1/Id3 KO) and Tek (Tie2)-rtTA, TRE-lacz/TRE Id1 (TRE Id1) mice with doxycycline inducible endothelial Id1 and β-galactosidase expression. Id1 and 3 were co-localized in endothelial cells in normal adult kidneys and protein levels were increased at day 3 following ischemia-reperfusion injury (IRI) and contralateral nephrectomy. Id1/Id3 KO mice had decreased baseline capillary density and pericyte coverage and increased tubular damage following IRI but decreased interstitial cell proliferation and fibrosis compared with WT littermates. No compensatory increase in kidney size occurred in KO mice resulting in increased creatinine compared with WT and TRE Id1 mice. TRE Id1 mice had no capillary rarefaction within 1 week following IRI in comparison with WT littermates. TRE Id1 mice had increased proliferation of PDGFRβ positive interstitial cells and medullary collagen deposition and developed capillary rarefaction and albuminuria at later time points. These differences were associated with increased Angiopoietin 1 (Ang1) and decreased Ang2 expression in TRE Id1 mice. Examination of gene expression in microvascular cells isolated from WT, Id1/Id3 KO and TRE Id1 mice showed increased Ang1 and αSMA in Id1 overexpressing cells and decreased pericyte markers in cells from KO mice. These results suggest that increased Id levels following AKI result in microvascular remodeling associated with increased fibrosis.

Non-invasive Assessment of Microvascular and Endothelial Function

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of cardiovascular disease. Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. Percent capillary recruitment is assessed by dividing the increase in capillary density induced by postocclusive reactive hyperemia (postocclusive reactive hyperemia capillary density minus baseline capillary density), by the maximal capillary density (observed during passive venous occlusion). Percent perfused capillaries represents the proportion of all capillaries present that are perfused (functionally active), and is calculated by dividing postocclusive reactive hyperemia capillary density by the maximal capillary density. Both percent capillary recruitment and percent perfused capillaries reflect the number of functional capillaries. The forearm blood flow (FBF) technique provides accepted non-invasive measures of endothelial function: The ratio FBF(max)/FBF(base) is computed as an estimate of vasodilation, by dividing the mean of the four FBF(max) values by the mean of the four FBFbase values. Forearm vascular resistance at maximal vasodilation (FVR(max)) is calculated as the mean arterial pressure (MAP) divided by FBF(max). Both the capillaroscopy and forearm techniques are readily acceptable to patients and can be learned quickly. The microvascular and endothelial function measures obtained using the methodologies described in this paper may have future utility in clinical patient cardiovascular risk-reduction strategies. As we have published reports demonstrating that microvascular and endothelial dysfunction are found in initial stages of hypertension including prehypertension, microvascular and endothelial function measures may eventually aid in early identification, risk-stratification and prevention of end-stage vascular pathology, with its potentially fatal consequences.

Postprandial microvascular function deteriorates in parallel with gradual worsening of insulin sensitivity and glucose tolerance in men with the metabolic syndrome or type 2 diabetes

Hyperinsulinaemia-induced whole-body glucose uptake during a euglycaemic-hyperinsulinaemic clamp is partly mediated by increased capillary density. We hypothesised that physiological insulinaemia in response to a mixed meal may also enhance microvascular function, and that this may be impaired in insulin-resistant individuals and patients with type 2 diabetes. Twelve men with uncomplicated type 2 diabetes, 13 with metabolic syndrome and 12 age-matched healthy normoglycaemic controls, mean age 57 ± 6 years, underwent skin capillary video microscopy before and 60 and 120 min following a standardised mixed meal to measure baseline capillary density (BCD) and capillary density during post-occlusive peak reactive hyperaemia (PRH), also termed capillary recruitment. Oral glucose insulin sensitivity (Matsuda index) and postprandial hyperglycaemia (2 h AUC(glucose)) were calculated. Fasting BCD was similar among groups, but fasting PRH was lowest in diabetes (p < 0.05). Postprandially, both BCD and PRH increased in all groups (p < 0.001); however, the meal-related increase in BCD was significantly lower in diabetes and metabolic syndrome vs controls (both p < 0.05). At all time points, postprandial PRH was lower in both diabetes and metabolic syndrome vs controls (both p < 0.05). In pooled analysis, postprandial mean PRH correlated with Matsuda index (r = 0.386, p = 0.018) and inversely with 2 h AUC(glucose) (r = -0.336, p = 0.042). Gradual deterioration in meal-related capillary recruitment was paralleled by decreasing insulin sensitivity and postprandial hyperglycaemia, as assessed in healthy normoglycaemic men, men with the metabolic syndrome and those with type 2 diabetes. These findings suggest that in both impaired glucose tolerance and in overt diabetes microvascular dysfunction might contribute to postprandial dysglycaemia. ClinicalTrials.gov NCT00721552.

Obesity is associated with impaired endothelial function in the postprandial state

Adequate microvascular perfusion is essential for the regulation of tissue metabolism. Therefore, defects in microvascular function may play a role in obesity-associated insulin resistance. Steady-state hyperinsulinemia during a euglycemic hyperinsulinemic clamp stimulates endothelium-dependent vasodilation and capillary recruitment, which contribute to increased glucose uptake. These phenomena have been shown to be blunted in obesity. If insulin's effects on microcirculatory function indeed play a physiological role in regulating insulin-mediated glucose uptake, such effects should be demonstrable not only during steady-state hyperinsulinemia, but also after meal ingestion. We investigated whether similar responses occur after ingestion of a glucose load or a mixed meal. We examined the effects of a glucose drink, a mixed meal drink, or a control drink (water) on skin capillary density (i.e. baseline capillary density, hyperemic capillary recruitment, and density during venous congestion, using capillaroscopy) and skin endothelium-(in)dependent vasodilation (using laser-Doppler flowmetry with iontophoresis of acetylcholine and sodium nitroprusside) in 20 lean and 19 obese individuals. In lean individuals, neither the glucose nor the mixed meal drink induced a significant effect on capillary density or endothelium-(in)dependent vasodilation. Possibly this is related to the modest plasma insulin levels as compared to the insulin clamp. In obese individuals, the mixed meal drink, compared to the control drink, decreased baseline skin perfusion (P<0.05) and acetylcholine-mediated vasodilation (P<0.05), while no effect of the drinks on capillary density was found. Compared to lean individuals, obese individuals had impaired acetylcholine-mediated vasodilation after meal ingestion (P=0.02). The latter findings are consistent with impaired postprandial microvascular function in obesity.

Acute angiotensin II receptor blockade improves insulin-induced microvascular function in hypertensive individuals

Objective An effect of insulin that is crucial for stimulating glucose uptake is its ability to increase the number of perfused capillaries, and thereby enhance its own delivery, and that of glucose, to muscle cells. To unravel possible mechanisms involved in the insulin-sensitizing effects of angiotensin II receptor blockers (ARBs) in hypertensive individuals we investigated the effect of single-dose ARB administration on insulin-mediated microvascular perfusion in hypertensive individuals. Methods We examined the effects of ARB administration on hyperinsulinemia-associated capillary density by measuring baseline skin capillary density, capillary density during reactive hyperemia (hyperemic capillary recruitment), and capillary density during venous congestion in 17 hypertensive individuals in the basal state, during a hyperinsulinemic euglycemic clamp, and during a hyperinsulinemic clamp with acute ARB administration (600 mg irbesartan), acute calcium channel blockade (CCB; 10 mg felodipine ER), as a control for the reduction in blood pressure, or placebo. In addition, insulin sensitivity and blood pressure were measured. Results Compared to the basal state, hyperinsulinemia increased baseline capillary density (57.3 ± 6.8 vs. 60.3 ± 7.9 n/mm 2, P < 0.01), but not hyperemic capillary recruitment. ARB and CCB treatment induced similar blood pressure reductions. Compared to placebo, ARB, but not CCB, increased hyperinsulinemia-associated baseline capillary density (+ 2.3 ± 3.4 ( P = 0.02) and − 0.4 ± 4.4 n/mm 2, respectively). Hyperinsulinemia-associated hyperemic capillary recruitment was not altered by either treatment. Compared to placebo, neither ARB nor CCB treatment enhanced insulin sensitivity. Conclusions Acute ARB administration increases insulin-induced microvascular perfusion in mildly hypertensive individuals; this beneficial effect on microvascular perfusion was however not associated with increased insulin-mediated glucose uptake.

Angiotensin II Enhances Insulin-Stimulated Whole-Body Glucose Disposal but Impairs Insulin-Induced Capillary Recruitment in Healthy Volunteers

Angiotensin II (AngII) increases insulin-mediated glucose uptake in healthy individuals. The underlying mechanisms are undefined. AngII may increase glucose uptake through a direct effect on muscle cell insulin signaling or through increasing insulin delivery to muscle cells through effects on the microvasculature. Our objective was to determine whether AngII increases insulin-mediated glucose uptake through effects on insulin-induced capillary recruitment. We examined the effects of AngII on hyperinsulinemia-induced capillary density by measuring skin capillary density, capillary recruitment, and capillary density during venous congestion in 18 healthy subjects in the basal state, during systemic hyperinsulinemia, and during hyperinsulinemia with coinfusion of AngII or phenylephrine (pressor control). In addition, whole-body glucose uptake and blood pressure were measured. Capillaroscopy data of 13 subjects were available for analysis. Compared with the basal state, hyperinsulinemia increased baseline capillary density (51.5+/-9.0 vs. 55.2+/-10.8 n/mm2, P<0.01), capillary recruitment (67.8+/-6.8 vs. 70.6+/-7.5 n/mm2, P<0.05), and capillary density during venous congestion (78.5+/-12.0 vs. 80.3+/-12.0 n/mm2, P<0.01). Infusion of AngII, but not phenylephrine, reduced insulin-induced capillary recruitment (69.3+/-8.6 vs. 65.2+/-8.0 n/mm2, P<0.05) and capillary density during venous congestion (79.7+/-15.3 vs. 73.9+/-12.1, P<0.05) while enhancing glucose uptake [2.40+/-0.7 vs. 2.68+/-0.6 (mg/kg.min per pmol/l)x100, P<0.01)] (n=18). AngII increases insulin-mediated glucose uptake in healthy individuals. This increase was probably not related to increases in microvascular perfusion because infusion of AngII during hyperinsulinemia reduced insulin-mediated skin capillary recruitment. Additional studies are needed to investigate whether AngII directly affects insulin delivery through increasing insulin transport across the microvasculature.

Abstract 3595: Chloride Intracellular Channel-4 Determines Native Collateral Density

The density of native (pre-existing) collaterals in healthy tissue and their capacity to enlarge in occlusive disease (arteriogenesis) are major determinants of tissue injury in ischemia. Mechanisms regulating arteriogenesis remain unclear. Moreover, nothing is known about what specifies formation of native collaterals, except our report that VEGF contributes to native collateral formation (Clayton et al FASEB J 21:LB352, 2007). Recently, chloride intracellular channel proteins (CLICs) have been implicated in cellular processes including cell hollowing. CLIC1 and CLIC4 are expressed by endothelial cells, and VEGF-induced cell hollowing is critical for vascular formation in the embryo and angiogenesis in ischemia. Therefore, we examined CLIC1−/− and CLIC4−/− (null) mice for effects on native collateral density in healthy tissues and on arteriogenesis and angiogenesis in a hindlimb model of ischemia. Responses of CLIC1−/−mice did not differ from wild-type in any experiment. In contrast, CLIC4−/− showed a greater reduction in perfusion immediately after femoral ligation (p&lt;0.05 here and all statements below). Since baseline capillary density and collateral diameter were not different from wild-type, this suggested CLIC4−/− have reduced native collateral density. This was confirmed angiographically. In agreement, during 10 days after ligation CLIC4−/− mice had worse recovery of perfusion, greater ischemia and atrophy, and more impaired limb use, whereas collateral remodeling was unaffected. While ischemia increased expression of CLIC1 and CLIC4 similarly in wild-type mice, CLIC1 was augmented 3-fold in CLIC4−/−, suggesting CLIC1 may compensate for absence of CLIC4. Despite greater ischemia in CLIC4−/− mice, HIF1α and VEGF increased less in CLIC4−/−, suggesting CLIC4 may reside upstream of Hif1α-VEGF signaling. Native collateral density in the cerebral circulation of CLIC4−/− mice (but not CLIC1−/−) was also greatly reduced, resulting in 4-fold larger infarctions. This reduced collateral density in adults was associated with reduced formation and stabilization of nascent collaterals in the newborn. Hence, CLIC4 represents the second gene identified, besides VEGF, that specifies formation of the native collateral circulation.

Effect of Electrical Stimulation on Arteriogenesis and Angiogenesis after Bilateral Femoral Artery Excision in the Rabbit Hind-Limb Ischemia Model

The effects of electrical stimulation (ES) on arteriogenesis (the opening of preexisting collaterals) and angiogenesis (formation of new capillaries) were studied after acute bilateral hind limb ischemia was induced via bilateral femoral artery excision in a rabbit model. The study evaluated the rabbit hind limbs' normal response to acute ischemia and to application of ES by calculating changes in arterial and capillary densities. Comparisons were made with our prior study, in which the femoral artery was unilaterally excised, as we attempted to expand on the topics of arteriogenesis and angiogenesis. Twelve adult New Zealand white rabbits were randomly assigned to 1 of 2 series. In Series 1, the control group, both femoral arteries were excised and no ES was applied. In Series 2, both femoral arteries were excised and ES was applied to the left limb. One lead was implanted into the left adductor muscle near the site of the excised left femoral artery (Series 2), and a stimulator (Thera, Medtronic, Inc, Minneapolis, MN) was implanted in a separate pocket. ES was applied at a rate of 3 V, 30 contractions per minute, beginning immediately after surgery and continuously for 1 month. Angiography was performed in all 12 rabbits 1 month after surgery to establish the anatomy of the collateral vessels and to demonstrate that the femoral artery stump continued to be an end artery. Contrast-opacified arteries (COAs) that crossed the grid's midline, and the total number of grid lines intersected by COAs, were tallied according to an established method. Capillary density was calculated as the number of capillaries per square millimeter of muscle. In Series 1, after 1 month, the number of COAs crossing the grid's midline was 4.5 +/-1.5 on the left and 4.8 +/-1.2 on the right side. In Series 2, the number of COAs crossing the grid's midline was 7.9 +/-1.8 on the left side (p<0.05 vs Series 1) and 5.9 +/-1.6 on the right side of the same rabbit (p=NS vs Series 1). In Series 1, 36.7 +/-5.4 and 30.5 +/-7.7 total intersections were crossed by COAs on the left and right sides, respectively. In Series 2, total grid intersections crossed by COAs were 48.4 +/-8.5 and 47.5 +/-9.1 in the left and right sides, respectively (p<0.001 vs series 1). Baseline capillary density before femoral artery excision was 180.2 +/-21.3/mm(2). The capillary densities on the left sides were 94.2 +/-19.1 and 264.5 +/-7.6 in Series 1 and 2, respectively (p<0.001). The right sides showed a similar pattern with capillary densities of 88.5 +/-37.2 and 135.8 +/-6.8 (p<0.05) in Series 1 and 2, respectively. When capillary density was compared on the left and right sides of the same rabbit in Series 2, a statistically significant increase was also found; 264.5 +/-7.6 vs 135.8 +/-6.8 (p<0.001) in the left and right sides, respectively. Comparisons of the effect of electrical stimulation and the body's normal physiologic response to acute ischemia revealed a significant increase in the opening of preexisting collaterals (arteriogenesis) and the promotion of capillary density (angiogenesis) with the use of electrical stimulation.

Maximization of skin capillaries during intravital video-microscopy in essential hypertension: comparison between venous congestion, reactive hyperaemia and core heat load tests

Intravital capillary video-microscopy is a dynamic method for studying skin capillaries. The technique of direct intravital microscopy (without dyes) depends on the presence of red blood cells inside capillaries for their identification. The aim of the present study was to compare different techniques to try to establish the best method for maximizing the number of visible perfused capillaries during intravital capillary microscopy. We compared the effects of venous congestion with those of post-occlusive reactive hyperaemia (Study 1). We also investigated venous congestion followed first by post-occlusive reactive hyperaemia and then by a core heat load test (Study 2). Finally we investigated venous congestion followed by post-occlusive reactive hyperaemia combined with venous congestion (Study 3). In Study 1, capillary density increased with venous congestion from a baseline value of 74+/-2 (mean+/-S.E.M.) per field to 82+/-3 per field (P<0.0001; analysis of variance). With reactive hyperaemia, there was an apparent decrease in visible capillary density to 69+/-2 per field. In Study 2, baseline capillary density was 69+/-4 per field, and this increased significantly with venous congestion to 74+/-4 per field (P=0.01). With both reactive hyperaemia and core heat load, the apparent density was 62+/-4 per field. In Study 3 the baseline density was 70+/-2 per field, and this increased significantly with venous congestion to 80+/-3 per field (P<0.0001). With reactive hyperaemia combined with venous congestion, the density was 81+/-3 per field (P=0.328 compared with venous congestion alone). The results show that venous congestion at 60 mmHg for 2 min is the most effective method for visualization of the maximal number of perfused skin capillaries during intravital video-microscopy.