Judith Brands

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.

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 ≤90 minutes after heparin as reflected by a decrease (P=0.008) in t50 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, t50 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.

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.

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 (10−5m) and sodium nitroprusside (10−6m) 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 μm during baseline to 0.17 ± 0.01 μm with bradykinin and 0.15 ± 0.01 μm 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 μm; 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.

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.

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.

Role for glycocalyx perturbation in atherosclerosis development and associated microvascular dysfunction

The importance of the endothelial glycocalyx for vascular homeostasis is becoming more and more evident. This review addresses the potential relation between a damaged glycocalyx and the process of atherosclerosis, including the associated impairment in blood-flow regulation. We envision restoration of glycocalyx perturbation in the future as a potential therapy for early treatment of cardiovascular disease.

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.

Modulation of Pre-Capillary Arteriolar Pressure with Drag-Reducing Polymers: A Novel Method for Enhancing Microvascular Perfusion

Please cite this paper as: Pacella JJ, Kameneva MV, Brands J, Lipowsky HH, Vink H, Lavery LL, Villanueva FS. Modulation of pre‐capillary arteriolar pressure with drag‐reducing polymers: a novel method for enhancing microvascular perfusion. Microcirculation 19: 580–585, 2012.

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.