Antiviral CD8+ T-cell immune responses are impaired by cigarette smoke and in COPD.

BACKGROUND: Virus infections drive COPD exacerbations and progression. Antiviral immunity centres on the activation of virus-specific CD8+ T-cells by viral epitopes presented on major histocompatibility complex (MHC) class I molecules of infected cells. These epitopes are generated by the immunoproteasome, a specialised intracellular protein degradation machine, which is induced by antiviral cytokines in infected cells. METHODS: We analysed the effects of cigarette smoke on cytokine- and virus-mediated induction of the immunoproteasome in vitro, ex vivo and in vivo using RNA and Western blot analyses. CD8+ T-cell activation was determined in co-culture assays with cigarette smoke-exposed influenza A virus (IAV)-infected cells. Mass-spectrometry-based analysis of MHC class I-bound peptides uncovered the effects of cigarette smoke on inflammatory antigen presentation in lung cells. IAV-specific CD8+ T-cell numbers were determined in patients' peripheral blood using tetramer technology. RESULTS: Cigarette smoke impaired the induction of the immunoproteasome by cytokine signalling and viral infection in lung cells in vitro, ex vivo and in vivo. In addition, cigarette smoke altered the peptide repertoire of antigens presented on MHC class I molecules under inflammatory conditions. Importantly, MHC class I-mediated activation of IAV-specific CD8+ T-cells was dampened by cigarette smoke. COPD patients exhibited reduced numbers of circulating IAV-specific CD8+ T-cells compared to healthy controls and asthmatics. CONCLUSION: Our data indicate that cigarette smoke interferes with MHC class I antigen generation and presentation and thereby contributes to impaired activation of CD8+ T-cells upon virus infection. This adds important mechanistic insight on how cigarette smoke mediates increased susceptibility of smokers and COPD patients to viral infections.

l-Citrulline supplementation during pregnancy improves perinatal and postpartum maternal vascular function in a mouse model of preeclampsia.

Preeclampsia is a spontaneously occurring pregnancy complication diagnosed by new-onset hypertension and end-organ dysfunction with or without proteinuria. This pregnancy-specific syndrome contributes to maternal morbidity and mortality and can have detrimental effects on fetal outcomes. Preeclampsia is also linked to increased risk of maternal cardiovascular disease throughout life. Despite intense investigation of this disorder, few treatment options are available. The aim of this study was to investigate the potential therapeutic effects of maternal l-citrulline supplementation on pregnancy-specific vascular dysfunction in the male C57BL/6J × female C57BL/6J C1q-/- preeclampsia-like mouse model. l-Citrulline is a nonessential amino acid that is converted to l-arginine to promote smooth muscle and blood vessel relaxation and improve nitric oxide (NO)-mediated vascular function. To model a preeclampsia-like pregnancy, female C57BL/6J mice were mated to C1q-/- male mice, and a subset of dams was supplemented with l-citrulline throughout pregnancy. Blood pressure, systemic vascular glycocalyx, and ex vivo vascular function were investigated in late pregnancy, and postpartum at 6 and 10 mo of age. Main findings show that l-citrulline reduced blood pressure, increased vascular glycocalyx volume, and rescued ex-vivo vascular function at gestation day 17.5 in this preeclampsia-like model. The vascular benefit of l-citrulline also extended postpartum, with improved vascular function and glycocalyx measures at 6 and 10 mo of age. l-Citrulline-mediated vascular improvements appear, in part, attributable to NO pathway signaling. Taken together, l-citrulline supplementation during pregnancy appears to have beneficial effects on maternal vascular health, which may have translational implications for improved maternal cardiovascular health.

Paternal deficiency of complement component C1q leads to a preeclampsia-like pregnancy in wild-type female mice and vascular adaptations postpartum.

Preeclampsia is a spontaneously occurring, pregnancy-specific syndrome that is clinically diagnosed by new onset hypertension and proteinuria. Epidemiological evidence describes an association between a history of preeclampsia and increased risk for cardiovascular disease in later life; however, the mechanism(s) driving this relationship are unclear. Our study aims to leverage a novel preeclampsia-like mouse model, the C1q-/- model, to help elucidate the acute and persistent vascular changes during and following a preeclampsia-like pregnancy. Female C57BL/6J mice were mated to C1q-/- male mice to model a preeclampsia-like pregnancy ("PE-like"), and the maternal cardiovascular phenotype (blood pressure, renal function, systemic glycocalyx, and ex vivo vascular function) was assessed in late pregnancy and postpartum at 6 and 10 mo of age. Uncomplicated, normotensive pregnancies (female C57BL/6J bred to male C57BL/6J mice) served as age-matched controls. In pregnancy, PE-like dams exhibited increased systolic and diastolic pressure during mid- and late gestation, renal dysfunction, fetal growth restriction, and reduced placental efficiency. Ex vivo wire myography studies of mesenteric arteries revealed severe pregnancy-specific endothelial-dependent and -independent vascular dysfunction. At 3 and 7 mo postpartum (6 and 10 mo old, respectively), hypertension resolved in PE-like dams, whereas mild vascular dysfunction persisted at 3 mo postpartum. In conclusion, the female C57BL/6J-by-male C57BL/6J C1q-/- model recapitulates many aspects of the human preeclampsia syndrome in a low-risk, wild-type female mouse. The pregnancy-specific phenotype results in systemic maternal endothelial-dependent and -independent vascular dysfunction that persists postpartum.

Association of Hypertensive Disorders of Pregnancy With Coronary Microvascular Dysfunction 8 to 10 Years After Delivery.

BACKGROUND: Hypertensive disorders of pregnancy (HDP) are associated with subsequent adverse cardiac remodeling and cardiovascular disease. The role of myocardial microvascular disease among individuals with HDP and left ventricular (LV) remodeling as a potential link to cardiovascular disease is unknown. We aimed to determine whether individuals with HDP history have coronary microvascular dysfunction measured by coronary flow reserve 8 to 10 years after delivery and whether microvascular dysfunction correlates with LV remodeling. METHODS: Individuals with pregnancies delivered from 2008 to 2010 underwent burst-replenishment myocardial contrast echocardiography (2017-2020) to quantify myocardial perfusion at rest and during dobutamine stress. Video intensity versus time data were used to derive ß, the rate of rise of video intensity, a correlate for myocardial blood flow. Coronary flow reserve was calculated as the ratio of ß at peak stress to ß at rest, averaged across LV myocardial regions of interest. RESULTS: We studied 91 individuals (aged 38±6 and 9.1±0.9 years postdelivery) and 19 with a history of HDP. Individuals with coronary microvascular dysfunction (coronary flow reserve <2.0; n=13) had a higher proportion of HDP (46.2% versus 16.7%; P=0.026) and higher prepregnancy body mass index, baseline heart rate, and hemoglobin A1c compared with those without microvascular dysfunction. The association of coronary flow reserve and HDP was attenuated after adjusting for cardiometabolic factors (P=0.133). In exploratory subgroup analyses, individuals with both LV remodeling (relative wall thickness >0.42) and HDP (n=12) had the highest proportion of microvascular dysfunction (41.7% versus +HDP-LV remodeling [n=7] 14.3%; -HDP+LV remodeling [n=26] 7.7%; P=0.0498). CONCLUSIONS: In this small study, HDP history is associated with coronary microvascular dysfunction 1 decade after delivery, findings that may, in part, be driven by metabolic factors including obesity and diabetes. Microvascular dysfunction may contribute to cardiovascular disease among individuals with a history of HDP.

CYB5R3 in type II alveolar epithelial cells protects against lung fibrosis by suppressing TGF-ß1 signaling.

Type II alveolar epithelial cell (AECII) redox imbalance contributes to the pathogenesis of idiopathic pulmonary fibrosis (IPF), a deadly disease with limited treatment options. Here, we show that expression of membrane-bound cytochrome B5 reductase 3 (CYB5R3), an enzyme critical for maintaining cellular redox homeostasis and soluble guanylate cyclase (sGC) heme iron redox state, is diminished in IPF AECIIs. Deficiency of CYB5R3 in AECIIs led to sustained activation of the pro-fibrotic factor TGF-ß1 and increased susceptibility to lung fibrosis. We further show that CYB5R3 is a critical regulator of ERK1/2 phosphorylation and the sGC/cGMP/protein kinase G axis that modulates activation of the TGF-ß1 signaling pathway. We demonstrate that sGC agonists (BAY 41-8543 and BAY 54-6544) are effective in reducing the pulmonary fibrotic outcomes of in vivo deficiency of CYB5R3 in AECIIs. Taken together, these results show that CYB5R3 in AECIIs is required to maintain resilience after lung injury and fibrosis and that therapeutic manipulation of the sGC redox state could provide a basis for treating fibrotic conditions in the lung and beyond.

Modulation of pre-capillary arteriolar pressure with drag-reducing polymers: a novel method for enhancing microvascular perfusion.

OBJECTIVE: We have shown that drag-reducing polymers (DRP) enhance capillary perfusion during severe coronary stenosis and increase red blood cell velocity in capillaries, through uncertain mechanisms. We hypothesize that DRP decreases pressure loss from the aorta to the arteriolar compartment. METHODS: Intravital microscopy of the rat cremaster muscle and measurement of pressure in arterioles (diameters 20-132 µm) was performed in 24 rats. DRP (polyethylene oxide, 1 ppm) was infused i.v. and measurements were made at baseline and 20 minutes after completion of DRP infusion. In a 10-rat subset, additional measurements were made three minutes after the start, and one to five and 10 minutes after completion of DRP. RESULTS: Twenty minutes after the completion of DRP, mean arteriolar pressure was 22% higher than baseline (from 42 ± 3 to 49 ± 3 mmHg, p < 0.005, n = 24). DRP decreased the pressure loss from the aorta to the arterioles by 24% (from 35 ± 6 to 27 ± 5 mmHg, p = 0.001, n = 10). In addition, there was a strong trend toward an increase in pressure at 10 minutes after the completion of DRP (n = 10). CONCLUSIONS: Drag-reducing polymers diminish pressure loss between the aorta and the arterioles. This results in a higher pre-capillary pressure and probably explains the observed DRP enhancement in capillary perfusion.

Maternal Vascular Lesions in the Placenta Predict Vascular Impairments a Decade After Delivery.

Women with adverse pregnancy outcomes later experience excess hypertension and cardiovascular disease, but how the events are linked is unknown. Examination of the placenta may provide clues to vascular impairments after delivery. Maternal vascular malperfusion lesions (MVMs) were abstracted from clinical reports, validated and characterized using clinical guidelines and severity score. A total of 492 women (170 with MVMs and 322 without MVMs) participated in a study visit 8 to 10 years after delivery to assess blood pressure, cardiometabolic factors, and sublingual microvascular features using sidestream dark field imaging. Covariates included age, race, adverse pregnancy outcomes (preeclampsia, small for gestational age, and preterm birth), and health behaviors. Women with versus without MVM had a distinct sublingual microvascular profile comprised of (1) lower microvascular density (-410 µm/mm2, P=0.015), (2) higher red blood cell filling as a marker of perfusion (2%, P=0.004), and (3) smaller perfused boundary region (-0.07 µm, P=0.025) as a measure of glycocalyx integrity, adjusted for covariates including adverse pregnancy outcomes. Women with MVM also had higher adjusted diastolic blood pressure (+2.6 mm Hg, P=0.021), total and LDL (low-density lipoprotein)-cholesterol (+11.2 mg/dL, P=0.016; +8.7 mg/dL, P=0.031). MVM associations with subsequent cardiovascular measures did not vary by type of adverse pregnancy outcome, except among women with preterm births where blood pressure was higher only among those with MVM. Results were similar when evaluated as MVM severity. A decade after delivery, women with placental vascular lesions had an adverse cardiovascular profile comprised of microvascular rarefaction, higher blood pressure and more atherogenic lipids. Placental histopathology may reveal a woman's early trajectory toward subsequent vascular disease.

Acute attenuation of glycocalyx barrier properties increases coronary blood volume independently of coronary flow reserve.

Vascular endothelium is covered with an extensive mesh of glycocalyx constituents, which acts like an effective barrier up to several micrometers thick that shields the luminal surface of the vasculature from direct exposure to flowing blood. Many studies report that various enzymatic and pharmaceutical challenges are able to increase glycocalyx porosity, resulting in farther permeation of plasma macromolecules and greater access of red blood cells into glycocalyx domain. Attenuation of glycocalyx barrier properties therefore potentially increases the amount of blood that effectively occupies available microvascular volume. We tested in the present study whether attenuation of coronary glycocalyx barrier properties actually increases coronary blood volume and whether such changes would be noticeable during measurements of coronary flow reserve using adenosine. In anesthetized goats (n = 6) with cannulated left main coronary artery that were perfused under controlled pressure, coronary blood volume was measured via the indicator-dilution technique using high-molecular-weight (2,000 kDa) dextrans as plasma tracer and labeled red blood cells as red blood cell tracer. Coronary blood volume was determined at baseline and during intracoronary infusion of adenosine causing maximal vasodilation (0.2-0.6 mg.kg(-1).h(-1)) before and after intracoronary hyaluronidase treatment (170,000 units) of the glycocalyx. With an intact glycocalyx, coronary blood volume was 18.9 +/- 1.1 ml/100 g heart tissue at baseline, which increased to 26.3 +/- 2.7 ml/100 g after hyaluronidase treatment of the coronary glycocalyx. Maximal vasodilation by administration of adenosine further increased coronary blood volume to 33.9 +/- 6.8 ml/100 g, a value not different from the maximal coronary blood volume of 33.2 +/- 5.3 ml/100 g obtained by administration of adenosine in the absence of hyaluronidase treatment. Adenosine-induced increases in coronary conductance were not affected by hyaluronidase treatment. We conclude that acute attenuation of glycocalyx barrier properties increases coronary blood volume by approximately 40%, which is of similar magnitude as additional changes in coronary blood volume during subsequent maximal vasodilation with adenosine. Furthermore, maximal coronary blood volume following administration of adenosine was similar with and without prior hyaluronidase degradation of the glycocalyx, suggesting that adenosine and hyaluronidase potentially increase glycocalyx porosity to a similar extent. Hyaluronidase-mediated changes in coronary blood volume did not affect baseline and adenosine-induced increases in coronary conductance, demonstrating that measurements of coronary flow reserve are insufficient to detect impairment of coronary blood volume recruitment in conditions of damaged glycocalyx.

Noninvasive sublingual microvascular imaging reveals sex-specific reduction in glycocalyx barrier properties in patients with coronary artery disease.

BACKGROUND: Risk factors for coronary artery disease (CAD) have been associated with endothelial dysfunction and degradation of the endothelial glycocalyx. This study was designed to compare sublingual microvascular perfusion and glycocalyx barrier properties in CAD patients and controls using noninvasive side stream darkfield imaging. METHODS: Imaging of the sublingual microvasculature was performed in 52 case subjects (CAD confirmed by left heart catheterization) and 63 controls (low Framingham risk score). Red blood cell (RBC) filling percentage and functional microvascular density, measures of microvascular perfusion, and perfused boundary region (PBR), an index of glycocalyx barrier function, were measured in microvessels with a diameter ranging from 5-25 µm. RESULTS: RBC filling percentage was lower in patients with CAD compared to controls (p < .001). Functional microvascular density did not differ between groups. The overall PBR was marginally greater in the CAD group compared to the control group (p = .08). PBR did not differ between male CAD cases and controls (p = .17). However, PBR was greater in females with CAD compared with female controls (p = .04), indicating reduced glycocalyx barrier function. This difference became more pronounced after adjusting for potential confounders. CONCLUSIONS: Our data suggest that patients with CAD are characterized by a reduction in percentage of time microvessels are occupied by RBCs. In addition, CAD is significantly associated with impaired sublingual microvascular glycocalyx barrier function in women but not men. More research is needed to determine the significance of peripheral microvascular dysfunction in the pathophysiology of CAD, and how this may differ by sex.

Bradykinin- and sodium nitroprusside-induced increases in capillary tube haematocrit in mouse cremaster muscle are associated with impaired glycocalyx barrier properties.

Previous studies have suggested that agonists may increase functionally perfused capillary volume by modulation of blood-excluding glycocalyx volume, but direct evidence for this association is lacking at the moment. Using intravital microscopic visualization of mouse cremaster muscle, we determined the effects of bradykinin (10(-5) M) and sodium nitroprusside (10(-6) M) on capillary tube haematocrit and glycocalyx barrier properties. In control C57Bl/6 mice (n = 10), tube haematocrit in capillaries (n = 71) increased (P < 0.05) from 8.7 +/- 0.3% during baseline to 21.2 +/- 1.2 and 22.2 +/- 0.9% during superfusion with bradykinin and nitroprusside, respectively. In parallel, the exclusion zone of FITC-labelled 70 kDa dextrans decreased (P < 0.05) from 0.37 +/- 0.01 microm during baseline to 0.17 +/- 0.01 microm with bradykinin and 0.15 +/- 0.01 microm with nitroprusside. Bradykinin and nitroprusside had no effect on dextran exclusion and tube haematocrit in capillaries (n = 55) of hyperlipidemic ApoE3-Leiden mice, which showed impaired exclusion of 70 kDa dextrans (0.05 +/- 0.02 microm; P < 0.05 versus C57Bl/6) and increased capillary tube haematocrit (23 +/- 0.8%; P < 0.05 versus C57Bl/6) under baseline conditions, indicating glycocalyx degradation. Our data show that vasodilator substances increase functionally perfused capillary volume and that this effect is associated with a reduction in glycocalyx exclusion of 70 kDa dextrans. Modulation of glycocalyx volume might represent a novel mechanism of perfusion control at the capillary level.

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.

ATF3 represses PINK1 gene transcription in lung epithelial cells to control mitochondrial homeostasis.

PINK1 (PTEN-induced putative kinase 1) is a key regulator of mitochondrial homeostasis that is relatively depleted in aging lungs and in lung epithelial cells from patients with idiopathic pulmonary fibrosis (IPF), a disease linked with aging. Impaired PINK1 expression and accumulation of damaged mitochondria in lung epithelial cells from fibrotic lungs were associated with the presence of ER stress. Here, we show that ATF3 (activating transcription factor 3), a member of the integrated stress response (ISR), negatively regulates transcription of the PINK1 gene. An ATF3 binding site within the human PINK1 promoter is located in the first 150 bp upstream of the transcription start site. Induction of ER stress or overexpression of ATF3 inhibited the activity of the PINK1 promoter. Importantly, overexpression of ATF3 causes accumulation of depolarized mitochondria, increased production of mitochondrial ROS, and loss of cell viability. Furthermore, conditional deletion of ATF3 in type II lung epithelial cells protects mice from bleomycin-induced lung fibrosis. Finally, we observed that ATF3 expression increases in the lung with age and, specially, in lung epithelial cells from IPF lungs. These data provide a unique link between ATF3 and PINK1 expression suggesting that persistent stress, driven by ATF3, can dysregulate mitochondrial homeostasis by repression of PINK1 mRNA synthesis.

Treatment of microvascular micro-embolization using microbubbles and long-tone-burst ultrasound: an in vivo study.

Despite epicardial coronary artery reperfusion by percutaneous coronary intervention, distal micro-embolization into the coronary microcirculation limits myocardial salvage during acute myocardial infarction. Thrombolysis using ultrasound and microbubbles (sonothrombolysis) is an approach that induces microbubble oscillations to cause clot disruption and restore perfusion. We sought to determine whether this technique could restore impaired tissue perfusion caused by thrombotic microvascular obstruction. In 16 rats, an imaging transducer was placed on the biceps femoris muscle, perpendicular to a single-element 1-MHz treatment transducer. Ultrasound contrast perfusion imaging was performed at baseline and after micro-embolization. Therapeutic ultrasound (5000 cycles, pulse repetition frequency = 0.33 Hz, 1.5 MPa) was delivered to nine rats for two 10-min sessions during intra-arterial infusion of lipid-encapsulated microbubbles; seven control rats received no ultrasound-microbubble therapy. Ultrasound contrast perfusion imaging was repeated after each treatment or control period, and microvascular volume was measured as peak video intensity. There was a 90% decrease in video intensity after micro-embolization (from 8.6 ± 4.8 to 0.7 ± 0.8 dB, p < 0.01). The first and second ultrasound-microbubble sessions were respectively followed by video intensity increases of 5.8 ± 5.1 and 8.7 ± 5.7 dB (p < 0.01, compared with micro-embolization). The first and second control sessions, respectively, resulted in no significant increase in video intensity (2.4 ± 2.3 and 3.6 ± 4.9) compared with micro-embolization (0.6 ± 0.7 dB). We have developed an in vivo model that simulates the distal thrombotic microvascular obstruction that occurs after primary percutaneous coronary intervention. Long-pulse-length ultrasound with microbubbles has a therapeutic effect on microvascular perfusion and may be a valuable adjunct to reperfusion therapy for acute myocardial infarction.

PINK1 deficiency impairs mitochondrial homeostasis and promotes lung fibrosis.

Although aging is a known risk factor for idiopathic pulmonary fibrosis (IPF), the pathogenic mechanisms that underlie the effects of advancing age remain largely unexplained. Some age-related neurodegenerative diseases have an etiology that is related to mitochondrial dysfunction. Here, we found that alveolar type II cells (AECIIs) in the lungs of IPF patients exhibit marked accumulation of dysmorphic and dysfunctional mitochondria. These mitochondrial abnormalities in AECIIs of IPF lungs were associated with upregulation of ER stress markers and were recapitulated in normal mice with advancing age in response to stimulation of ER stress. We found that impaired mitochondria in IPF and aging lungs were associated with low expression of PTEN-induced putative kinase 1 (PINK1). Knockdown of PINK1 expression in lung epithelial cells resulted in mitochondria depolarization and expression of profibrotic factors. Moreover, young PINK1-deficient mice developed similarly dysmorphic, dysfunctional mitochondria in the AECIIs and were vulnerable to apoptosis and development of lung fibrosis. Our data indicate that PINK1 deficiency results in swollen, dysfunctional mitochondria and defective mitophagy, and promotes fibrosis in the aging lung.

Effect of acute hyaluronidase treatment of the glycocalyx on tracer-based whole body vascular volume estimates in mice.

The endothelial glycocalyx forms a hyaluronan-containing interface between the flowing blood and the endothelium throughout the body. By comparing the systemic distribution of a small glycocalyx-accessible tracer vs. a large circulating plasma tracer, the size-selective barrier properties of the glycocalyx have recently been utilized to estimate whole body glycocalyx volumes in humans and animals, but a comprehensive validation of this approach has been lacking at the moment. In the present study, we compared, in anesthetized, ventilated C57Bl/6 mice, the whole body distribution of small (40 kDa) dextrans (Texas Red labeled; Dex40) vs. that of intermediate (70 kDa) and large (500 kDa) dextrans (both FITC labeled; Dex70 and Dex500, respectively) using tracer dilution and vs. that of circulating plasma, as derived from the dilution of fluorescein-labeled red blood cells and large-vessel hematocrit. The contribution of the glycocalyx was evaluated by intravenous infusion of a bolus of the enzyme hyaluronidase. In saline-treated control mice, distribution volume (in ml) differed between tracers (P < 0.05; ANOVA) in the following order: Dex40 (0.97 ± 0.04) > Dex70 (0.90 ± 0.04) > Dex500 (0.81 ± 0.10) > plasma (0.71 ± 0.02), resulting in an inaccessible vascular volume, i.e., compared with the distribution volume of Dex40, of 0.03 ± 0.01, 0.15 ± 0.04, and 0.31 ± 0.05 ml for Dex70, Dex500, and plasma, respectively. In hyaluronidase-treated mice, Dex70 and Dex40 volumes were not different from each other, and inaccessible vascular volumes for Dex500 (0.03 ± 0.03) and plasma (0.14 ± 0.05) were smaller (P < 0.05) than those in control animals. Clearance of Dex70 and Dex500 from the circulation was enhanced (P < 0.05) in hyaluronidase-treated vs. control mice. These results indicate that the glycocalyx contributes to size-dependent differences in whole body vascular distribution of plasma solutes in mice. Whole body vascular volume measurements based on the differential distribution of glycocalyx-selective tracers appear appropriate for the detection of generalized glycocalyx degradation in experimental animals and humans.

Whole-body recruitment of glycocalyx volume during intravenous adenosine infusion.

Adenosine-mediated recruitment of microvascular volume in heart and muscle has been suggested to include, in addition to vasodilation of resistance vessels, an increased accessibility of the endothelial glycocalyx for flowing plasma as a result of an impairment of its barrier properties. The aim of the current study was to investigate the effect of systemic intravenous administration of adenosine on the glycocalyx-dependent exclusion of circulating blood at a whole-body level. In anesthetized goats (N = 6), systemic blood-excluded glycocalyx volume was measured by comparing the intravascular distribution volume of the suggested glycocalyx accessible tracer dextrans with a molecular weight of 40 kDa (Dex-40) to that of circulating plasma, derived from the dilution of labeled red blood cells and large vessel hematocrit. Systemic glycocalyx volume was determined at baseline and during intravenous infusion of adenosine (157 ± 11.6 µg/kg min(-1)). Blood-inaccessible glycocalyx volume decreased from 458.1 ± 95.5 to 18.1 ± 62.2 mL (P < 0.01) during adenosine administration. While circulating plasma volume did not change significantly (617.1 ± 48.5 vs. 759.2 ± 47.9 mL, NS), the decrease in blood-excluded glycocalyx volume was associated with a decrease in Dex-40 distribution volume (from 1075.2 ± 71.0 to 777.3 ± 60.0 mL, P < 0.01). Intravenous administration of adenosine is associated with a robust impairment of whole-body glycocalyx barrier properties, reflected by a greatly reduced exclusion of circulating blood compared to small dextrans. The observed decrease in Dex-40 distribution volume suggests that the reduction in glycocalyx volume coincides with a reduction in tracer-accessible vascular volume.

New insights into the microvascular mechanisms of drag reducing polymers: effect on the cell-free layer.

Drag-reducing polymers (DRPs) significantly increase blood flow, tissue perfusion, and tissue oxygenation in various animal models. In rectangular channel microfluidic systems, DRPs were found to significantly reduce the near-wall cell-free layer (CFL) as well as modify traffic of red blood cells (RBC) into microchannel branches. In the current study we further investigated the mechanism by which DRP enhances microvascular perfusion. We studied the effect of various concentrations of DRP on RBC distribution in more relevant round microchannels and the effect of DRP on CFL in the rat cremaster muscle in vivo. In round microchannels hematocrit was measured in parent and daughter branch at baseline and after addition of DRP. At DRP concentrations of 5 and 10 ppm, the plasma skimming effect in the daughter branch was eliminated, as parent and daughter branch hematocrit were equivalent, compared to a significantly lowered hematocrit in the daughter branch without DRPs. In anesthetized rats (N=11) CFL was measured in the cremaster muscle tissue in arterioles with a diameter of 32.6 ± 1.7 µm. In the control group (saline, N=6) there was a significant increase in CFL in time compared to corresponding baseline. Addition of DRP at 1 ppm (N=5) reduced CFL significantly compared to corresponding baseline and the control group. After DRP administration the CFL reduced to about 85% of baseline at 5, 15, 25 and 35 minutes after DRP infusion was complete. These in vivo and in vitro findings demonstrate that DRPs induce a reduction in CFL width and plasma skimming in the microvasculature. This may lead to an increase of RBC flux into the capillary bed, and thus explain previous observations of a DRP mediated enhancement of capillary perfusion.

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.

Agonist-induced impairment of glycocalyx exclusion properties: contribution to coronary effects of adenosine.

The endothelial glycocalyx is the negatively charged, gel-like mesh residing at the luminal side of the vascular endothelium and forming the interface between the flowing blood and the vessel wall. The vast majority of glycocalyx volume resides in the microcirculation, particularly in the capillaries. Intravital microscopic observations of capillaries in striated muscle preparations illustrate that under resting conditions, the glycocalyx is not accessible for flowing red blood cells and greatly hinders plasma flow in the axial direction, causing a significant reduction in functionally perfused capillary volume. Glycocalyx exclusion properties have been shown to be reduced by adenosine and other vasoactive substances. A diminished exclusion of circulating blood by the glycocalyx may facilitate nutrient exchange since it is associated with an increase in functionally perfused blood volume and surface area in the capillaries. Our recent studies have focused on the effect of adenosine on glycocalyx exclusion in the coronary circulation and demonstrate an important role for this mechanism in the increase in circulating coronary blood volume during administration of this vasodilator. The current review elaborates on the glycocalyx as a blood-excluding intravascular layer and how it can be modulated by various agonists. Further, the potential role of adenosine-induced modulation of glycocalyx exclusion properties in coupling increases in blood flow and circulating blood volume in the coronary circulation is discussed. Finally, we consider how degradation of the glycocalyx may impact on coronary blood volume regulation, thereby providing new opportunities to diagnose glycocalyx damage in the clinical setting.

Heparin impairs glycocalyx barrier properties and attenuates shear dependent vasodilation in mice.

The endothelial glycocalyx is a hydrated mesh of polysaccharides and adsorbed plasma proteins that forms the true interface between the flowing blood and the endothelium. We hypothesized in the present study that competitive binding of heparin to glycocalyx-associated proteins would affect glycocalyx barrier properties and mechanotransduction of shear stress to the endothelium. In anesthetized mice, the clearance of 70-kDa dextrans from the circulation was increased (P<0.05 versus saline) 1 hour after heparin (1.25 U) and glycocalyx degradation with hyaluronidase (35 U; amount cleared in 30 minutes after saline: 11+/-5%; after heparin: 45+/-8%; after hyaluronidase: 30+/-3%). Clearance of 40-kDa dextrans increased (P<0.05 versus saline) to a lesser extent after both treatments (saline: 46+/-3%; heparin: 60+/-5%; hyaluronidase: 60+/-2%). The dilator response of second-order arterioles in cremaster muscle during reactive hyperemia was reduced for < or =90 minutes after heparin as reflected by a decrease (P=0.008) in t(50) of diameter recovery, and this effect was associated with a diminished NO bioavailability. Infusion of hyaluronidase resulted in reductions (P<0.05) in baseline and peak reactive hyperemic diameter, whereas, despite an increase in wall shear rate at the beginning of reactive hyperemia, t(50) of diameter recovery was not affected. In conclusion, our data in mice show that a heparin challenge is associated with increased vascular leakage of dextrans and impaired arteriolar vasodilation during reactive hyperemia. Our data suggest that protein-heparan sulfate interactions are important for a functional glycocalyx.