Metformin and sulodexide restore cardiac microvascular perfusion capacity in diet-induced obese rats.

BACKGROUND: Disturbances in coronary microcirculatory function, such as the endothelial glycocalyx, are early hallmarks in the development of obesity and insulin resistance. Accordingly, in the present study myocardial microcirculatory perfusion during rest and stress was assessed following metformin or sulodexide therapy in a rat model of diet-induced obesity. Additionally, the effect of degradation of the glycocalyx on myocardial perfusion was assessed in chow-fed rats. METHODS: Rats were fed a high fat diet (HFD) for 8 weeks and were divided into a group without therapy, and groups that received the anti-diabetic drug metformin or the glycocalyx-stabilizing drug sulodexide in their drinking water during the last 4 weeks of the feeding period. Myocardial microvascular perfusion was determined using first-pass perfusion MRI before and after adenosine infusion. The effect of HFD on microcirculatory properties was also assessed by sidestream darkfield (SDF) imaging of the gastrocnemius muscle. In an acute experimental setting, hyaluronidase was administered to chow-fed control rats to determine the effect of enzymatical degradation of the glycocalyx on myocardial perfusion. RESULTS: HFD-rats developed central obesity and insulin sensitivity was reduced as evidenced by the marked reduction in insulin-induced phosphorylation of Akt in both cardiac and gastrocnemius muscle. We confirmed our earlier findings that the robust increase in myocardial perfusion in chow-fed rats after an adenosine challenge (+56%, p = 0.002) is blunted in HFD rats (+8%, p = 0.68). In contrast, 4-weeks treatment with metformin or sulodexide partly restored the increase in myocardial perfusion during adenosine infusion in HFD rats (+81%, p = 0.002 and +37%, p = 0.02, respectively). Treating chow-fed rats acutely with hyaluronidase, to enzymatically degrade the glyocalyx, completely blunted the increase in myocardial perfusion during stress. CONCLUSIONS: In early stages of HFD-induced insulin resistance myocardial perfusion becomes compromised, a process that can be countered by treatment with both metformin and sulodexide. The adverse effect of acute glycocalyx degradation and protective effect of long-term sulodexide administration on myocardial perfusion provides indirect evidence, suggesting a role for the glycocalyx in preserving coronary microvascular function in pre-diabetic animals.

Perturbed mechanotransduction by endothelial surface glycocalyx modification greatly impairs the arteriogenic process.

The mechanisms that trigger initiation of arteriogenesis in response to pathogenic obstruction of arterial flow are not fully understood. Our objective is to determine whether glycocalyx mediated mechanotransduction of fluid shear stress to the endothelial layer is an essential first step in inducing arteriogenesis. Mice were implanted with an osmotic minipump containing saline or hyaluronan synthase inhibitor 4-methylesculetin (4ME) 2 wk before femoral artery ligation. 4ME was effective in modifying the endothelial glycocalyx as measured by dextran exclusion and perfused boundary region changes. Glycocalyx modification resulted in a 52% (P = 0.002) reduction in perfusion restoration through the 21-day follow-up [area under the curve, 4.9 ± 1.1 (n = 11) vs. 10.2 ± 3.2 (n = 10), 4ME vs. control (Ctrl)]. Upon femoral artery ligation, no change in collateral vessel diameter in 4ME treated mice (49.8 ± 26.3 vs. 47.1 ± 14.0 µm, ligated vs unligated) was observed (Ctrl, 88.5 ± 18.8 vs. 35.1 ± 3.0 µm, ligated vs unligated, P < 0.05). This impaired arteriogenic process was accompanied by lack of local induction of both endothelial and smooth muscle cell activation (Ki67, endothelial nitric oxide synthase, and ICAM-1), as well as a failure to recruit CD11b-positive cells in 4ME-treated collateral vessels (0.012 ± 0.003 vs. 0.010 ± 0.003 cells/µm vessel perimeter, ligated vs. unligated), whereas in Ctrls, the number of CD11b cells was increased (0.024 ± 0.002 vs. 0.010 ± 0.004 cells/µm vessel perimeter, P < 0.05). Modification of the glycocalyx by inhibition of hyaluronan synthesis renders the endothelium unresponsive to altered hemodynamic conditions resulting from femoral artery ligation, which results in a hampered restoration of distal perfusion.

Early impairment of coronary microvascular perfusion capacity in rats on a high fat diet.

BACKGROUND: It remains to be established if, and to what extent, the coronary microcirculation becomes compromised during the development of obesity and insulin resistance. Recent studies suggest that changes in endothelial glycocalyx properties contribute to microvascular dysfunction under (pre-)diabetic conditions. Accordingly, early effects of diet-induced obesity on myocardial perfusion and function were studied in rats under baseline and hyperaemic conditions. METHODS: Rats were fed a high fat diet (HFD) for 6 weeks and myocardial microvascular perfusion was determined using first-pass perfusion MRI before and after adenosine infusion. The effect of HFD on microcirculatory properties was also assessed by sidestream darkfield (SDF) imaging of the gastrocnemius muscle. RESULTS: HFD-fed rats developed central obesity and insulin sensitivity was reduced as evidenced by the marked reduction in insulin-induced phosphorylation of Akt in both cardiac and gastrocnemius muscle. Early diet-induced obesity did not lead to hypertension or cardiac hypertrophic remodeling. In chow-fed, control rats a robust increase in cardiac microvascular perfusion was observed upon adenosine infusion (+40%; p < 0.05). In contrast, the adenosine response was abrogated in rats on a HFD (+8%; N.S.). HFD neither resulted in rarefaction or loss of glycocalyx integrity in skeletal muscle, nor reduced staining intensity of the glycocalyx of cardiac capillaries. CONCLUSIONS: Alterations in coronary microcirculatory function as assessed by first-pass perfusion MRI represent one of the earliest obesity-related cardiac adaptations that can be assessed non-invasively. In this early stage of insulin resistance, disturbances in glycocalyx barrier properties appeared not to contribute to the observed changes in coronary microvascular function.

Effects of two weeks of metformin treatment on whole-body glycocalyx barrier properties in db/db mice.

BACKGROUND: The anti-diabetic drug metformin has been demonstrated to exert a protective effect against vascular complications in diabetes independent of its glucose lowering action. Since the endothelial glycocalyx has been indicated to have important vasculoprotective properties and to be vulnerable to degradation by hyperglycemic conditions, we evaluated in the current study the effect of short-term metformin treatment on whole-body glycocalyx barrier properties in a mouse model of non-insulin dependent diabetes mellitus (db/db mouse). METHODS: Glycocalyx barrier properties were measured in an acute experiment in three groups of mice: 1) db/db mice without treatment serving as controls, 2) db/db mice which received metformin for two weeks in the drinking water serving as experimental group, and 3) C57Bl/6 mice serving as reference group. Animals were put under anesthesia (ketamine, medetomidine, and atropine) and carotid artery blood pressure was continuously monitored. To probe the glycocalyx a mixture of the tracers FITC-labeled 70 kDa dextrans (Dex70) or fluorescein-labeled red blood cells (RBCs) versus Texas Red-labeled 40 kDa dextrans (Dex40) was infused and blood samples subsequently collected for 30 min to determine the initial vascular distribution volume and clearance of these tracers. Urine was collected and dry-to-wet weight of heart and kidney were determined after the experiment. Group differences were tested using unpaired t-tests. RESULTS: Metformin treatment did not affect body weight, fasting blood glucose and arterial blood pressure. Compared to C57Bl/6 mice, db/db mice showed a diminished initial exclusion and increased vascular clearance of Dex70 versus Dex40 (P < 0.05), and both were improved by the metformin treatment (P < 0.05). While urine production was higher in the db/db mice compared to C57Bl/6 (P < 0.05), heart and kidney of the metformin treated animals showed comparable dry-to-wet weights compared to the C57Bl/6 mice. CONCLUSIONS: Two weeks of metformin in the drinking water is associated with an improvement in glycocalyx barrier properties in db/db mice, as evidence by an enhanced exclusion and retention of 70 kDa dextrans in the vasculature. In addition, metformin improved hydration of heart and kidney. Previous reported cardiovascular benefits of metformin may well involve an improvement of the endothelial glycocalyx.

The aPC treatment improves microcirculation in severe sepsis/septic shock syndrome.

BACKGROUND: The role of recombinant activated protein C (aPC) during sepsis is still controversial. It showed anti-inflammatory effect and improved the microvascular perfusion in experimental models of septic shock. The present study was aimed at testing the hypothesis that recombinant aPC therapy improves the microcirculation during severe sepsis. METHODS: Prospective observational study on patients admitted in a 12-beds intensive care unit of a university hospital from July 2010 to December 2011, with severe sepsis and at least two sepsis-induced organ failures occurring within 48 hours from the onset of sepsis, who received an infusion of aPC (24 mcg/kg/h for 96 hours) (aPC group). Patients with contraindications to aPC administration were also monitored (no-aPC group).At baseline (before starting aPC infusion, T0), after 24 hours (T1a), 48 hours (T1b), 72 hours (T1c) and 6 hours after the end of aPC infusion (T2), general clinical and hemodynamic parameters were collected and the sublingual microcirculation was evaluated with sidestream dark-field imaging. Total vessel density (TVD), perfused vessel density (PVD), De Backer score, microvascular flow index (MFIs), the proportion of perfused vessels (PPV) and the flow heterogeneity index (HI) were calculated for small vessels. The perfused boundary region (PBR) was measured as an index of glycocalyx damage. Variables were compared between time points and groups using non parametric or parametric statistical tests, as appropriate. RESULTS: In the 13 aPC patients mean arterial pressure (MAP), base excess, lactate, PaO2/FiO2 and the Sequential Organ Failure Assessment (SOFA) score significantly improved over time, while CI and ITBVI did not change. MFIs, TVD, PVD, PPV significantly increased over time and the HI decreased (p < 0.05 in all cases), while the PBR did not change. No-aPC patients (n = 9) did not show any change in the microcirculation over time. A positive correlation was found between MFIs and MAP. TVD, PVD and De Backer score negatively correlated with norepinephrine dose, and the SOFA score negatively correlated with MFIs, TVD and PVD. CONCLUSIONS: aPC significantly improves the microcirculation in patients with severe sepsis/septic shock. TRIAL REGISTRATION: NCT01806428.

Endothelial glycocalyx: sweet shield of blood vessels.

At the time that the term glycocalyx ("sweet husk") was introduced as a description of the extracellular polysaccharide coating on cells (Bennett HS: 1963. Morphological aspects of extracellular polysaccharides. J Hist Cytochem 11:14-23.), early electron microscopic observations had shown that anionic polysaccharides were also presented by the inner surface of blood vessels but the length of these structures was considered to be small and their functional significance was unknown. Research in the past decades in the glycocalyx field has evolved, and recent estimations indicate that the endothelial glycocalyx constitutes a voluminous intravascular compartment that plays an important role in vascular wall homeostasis. Pathologic loss of glycocalyx may be associated with an impaired vascular wall protection throughout the circulatory system, whereas agonist-induced modulation of glycocalyx accessibility for circulating blood may constitute a physiologically relevant mechanism to regulate functionally perfused volume and exchange area at the microvascular level. Both aspects are discussed in the current review.

SPARC preserves endothelial glycocalyx integrity, and protects against adverse cardiac inflammation and injury during viral myocarditis.

Myocardial damage as a consequence of cardiotropic viruses leads to a broad variety of clinical presentations and is still a complicated condition to diagnose and treat. Whereas the extracellular matrix protein Secreted Protein Acidic and Rich in Cysteine or SPARC has been implicated in hypertensive and ischemic heart disease by modulating collagen production and cross-linking, its role in cardiac inflammation and endothelial function is yet unknown. Absence of SPARC in mice resulted in increased cardiac inflammation and mortality, and reduced cardiac systolic function upon coxsackievirus-B3 induced myocarditis. Intra-vital microscopic imaging of the microvasculature of the cremaster muscle combined with electron microscopic imaging of the microvasculature of the cardiac muscle uncovered the significance of SPARC in maintaining endothelial glycocalyx integrity and subsequent barrier properties to stop inflammation. Moreover, systemic administration of recombinant SPARC restored the endothelial glycocalyx and consequently reversed the increase in inflammation and mortality observed in SPARC KO mice in response to viral exposure. Reducing the glycocalyx in vivo by systemic administration of hyaluronidase, an enzyme that degrades the endothelial glycocalyx, mimicked the barrier defects found in SPARC KO mice, which could be restored by subsequent administration of recombinant SPARC. In conclusion, the secreted glycoprotein SPARC protects against adverse cardiac inflammation and mortality by improving the glycocalyx function and resulting endothelial barrier function during viral myocarditis.

Non-invasive assessment of microvascular dysfunction in patients with microvascular angina.

BACKGROUND: We aimed to evaluate the microvascular function in patients with microvascular angina (MVA) by assessing 1) the endothelial glycocalyx barrier properties using sublingual microscopy, and 2) the myocardial perfusion reserve using cardiovascular magnetic resonance (CMR) imaging. METHODS: Sublingual microscopy was performed in 13 MVA patients (angina pectoris, ST-depression on treadmill testing, normal coronary angiogram) and compared with 2 control groups of 13 volunteers and 14 patients with known obstructive coronary artery disease (CAD). To test the glycocalyx-mediated microvascular responsiveness, the erythrocyte perfused boundary region (PBR) was assessed at baseline and after nitroglycerin challenge. RESULTS: The baseline PBR of MVA patients was similar to controls with CAD (p=0.72), and larger than in volunteers (p=0.02). Only the volunteers demonstrated a significant increase in PBR after nitroglycerin (p=0.03). In the 13 MVA patients, adenosine stress CMR perfusion imaging was performed. Although a significant increase in myocardial perfusion was observed in both the subendocardium and subepicardium during stress, the subendocardial perfusion reserve was significantly lower (p=0.02). The PBR responsiveness of the sublingual microvasculature showed a strong correlation with the transmural myocardial perfusion reserve (r=0.86, p<0.001). CONCLUSIONS: Patients with MVA can be characterized by microvascular glycocalyx dysfunction using sublingual microscopy. The strong correlation between sublingual PBR responsiveness and myocardial perfusion reserve suggests that the glycocalyx may play an important role in the regulation of microvascular volume for myocardial perfusion and supports the concept of impaired glycocalyx barrier properties in MVA.

Deeper penetration of erythrocytes into the endothelial glycocalyx is associated with impaired microvascular perfusion.

Changes in endothelial glycocalyx are one of the earliest changes in development of cardiovascular disease. The endothelial glycocalyx is both an important biological modifier of interactions between flowing blood and the vessel wall, and a determinant of organ perfusion. We hypothesize that deeper penetration of erythrocytes into the glycocalyx is associated with reduced microvascular perfusion. The population-based prospective cohort study (the Netherlands Epidemiology of Obesity [NEO] study) includes 6,673 middle-aged individuals (oversampling of overweight and obese individuals). Within this cohort, we have imaged the sublingual microvasculature of 915 participants using sidestream darkfield (SDF) imaging together with a recently developed automated acquisition and analysis approach. Presence of RBC (as a marker of microvascular perfusion) and perfused boundary region (PBR), a marker for endothelial glycocalyx barrier properties for RBC accessibility, were assessed in vessels between 5 and 25 µm RBC column width. A wide range of variability in PBR measurements, with a mean PBR of 2.14 µm (range: 1.43-2.86 µm), was observed. Linear regression analysis showed a marked association between PBR and microvascular perfusion, reflected by RBC filling percentage (regression coefficient ß: -0.034; 95% confidence interval: -0.037 to -0.031). We conclude that microvascular beds with a thick ("healthy") glycocalyx (low PBR), reflects efficient perfusion of the microvascular bed. In contrast, a thin ("risk") glycocalyx (high PBR) is associated with a less efficient and defective microvascular perfusion.

Association of kidney function with changes in the endothelial surface layer.

BACKGROUND AND OBJECTIVES: ESRD is accompanied by endothelial dysfunction. Because the endothelial glycocalyx (endothelial surface layer) governs interactions between flowing blood and the vessel wall, perturbation could influence disease progression. This study used a novel noninvasive sidestream-darkfield imaging method, which measures the accessibility of red blood cells to the endothelial surface layer in the microcirculation (perfused boundary region), to investigate whether renal function is associated with endothelial surface layer dimensions. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Perfused boundary region was measured in control participants (n=10), patients with ESRD (n=23), participants with normal kidney function after successful living donor kidney transplantation (n=12), and patients who developed interstitial fibrosis/tubular atrophy after kidney transplantation (n=10). In addition, the endothelial activation marker angiopoietin-2 and shed endothelial surface layer components syndecan-1 and soluble thrombomodulin were measured using ELISA. RESULTS: Compared with healthy controls (1.82 ± 0.16 µm), ESRD patients had a larger perfused boundary region (+0.23; 95% confidence interval, 0.46 to <0.01; P<0.05), which signifies loss of endothelial surface layer dimensions. This large perfused boundary region was accompanied by higher circulating levels of syndecan-1 (+57.71; 95% confidence interval, 17.38 to 98.04; P<0.01) and soluble thrombomodulin (+12.88; 95% confidence interval, 0.29 to 25.46; P<0.001). After successful transplantation, the perfused boundary region was indistinguishable from healthy controls (without elevated levels of soluble thrombomodulin or syndecan-1). In contrast, however, patients who developed interstitial fibrosis and tubular atrophy showed a large perfused boundary region (+0.36; 95% confidence interval, 0.09 to 0.63; P<0.01) and higher levels of endothelial activation markers. In addition, a significant correlation between perfused boundary region, angiopoietin-2, and eGFR was observed (perfused boundary region versus GFR: Spearman's ρ=0.31; P<0.05; perfused boundary region versus angiopoietin-2: Spearman's ρ=-0.33; P<0.05). CONCLUSION: Reduced renal function is strongly associated with low endothelial surface layer dimensions. After successful kidney transplantation, the endothelial surface layer is indistinguishable from control.

Comparison of four mathematical models to analyze indicator-dilution curves in the coronary circulation.

While several models have proven to result in accurate estimations when measuring cardiac output using indicator dilution, the mono-exponential model has primarily been chosen for deriving coronary blood/plasma volume. In this study, we compared four models to derive coronary plasma volume using indicator dilution; the mono-exponential, power-law, gamma-variate, and local density random walk (LDRW) model. In anesthetized goats (N = 14), we determined the distribution volume of high molecular weight (2,000 kDa) dextrans. A bolus injection (1.0 ml, 0.65 mg/ml) was given intracoronary and coronary venous blood samples were taken every 0.5-1.0 s; outflow curves were analyzed using the four aforementioned models. Measurements were done at baseline and during adenosine infusion. Absolute coronary plasma volume estimates varied by ~25% between models, while the relative volume increase during adenosine infusion was similar for all models. The gamma-variate, LDRW, and mono-exponential model resulted in volumes corresponding with literature, whereas the power-model seemed to overestimate the coronary plasma volume. The gamma-variate and LDRW model appear to be suitable alternative models to the mono-exponential model to analyze coronary indicator-dilution curves, particularly since these models are minimally influenced by outliers and do not depend on data of the descending slope of the curve only.