Diabetic Microvascular Disease and Pulmonary Fibrosis: The Contribution of Platelets and Systemic Inflammation

Diabetes is strongly associated with systemic inflammation and oxidative stress, but its effect on pulmonary vascular disease and lung function has often been disregarded. Several studies identified restrictive lung disease and fibrotic changes in diabetic patients and in animal models of diabetes. While microvascular dysfunction is a well-known complication of diabetes, the mechanisms leading to diabetes-induced lung injury have largely been disregarded. We described the potential involvement of diabetes-induced platelet-endothelial interactions in perpetuating vascular inflammation and oxidative injury leading to fibrotic changes in the lung. Changes in nitric oxide synthase (NOS) activation and decreased NO bioavailability in the diabetic lung increase platelet activation and vascular injury and may account for platelet hyperreactivity reported in diabetic patients. Additionally, the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway has been reported to mediate pancreatic islet damage, and is implicated in the onset of diabetes, inflammation and vascular injury. Many growth factors and diabetes-induced agonists act via the JAK/STAT pathway. Other studies reported the contribution of the JAK/STAT pathway to the regulation of the pulmonary fibrotic process but the role of this pathway in the development of diabetic lung fibrosis has not been considered. These observations may open new therapeutic perspectives for modulating multiple pathways to mitigate diabetes onset or its pulmonary consequences

Ceftriaxone preserves glutamate transporters and prevents intermittent hypoxia-induced vulnerability to brain excitotoxic injury.

Hypoxia alters cellular metabolism and although the effects of sustained hypoxia (SH) have been extensively studied, less is known about chronic intermittent hypoxia (IH), commonly associated with cardiovascular morbidity and stroke. We hypothesize that impaired glutamate homeostasis after chronic IH may underlie vulnerability to stroke-induced excitotoxicity. P16 organotypic hippocampal slices, cultured for 7 days were exposed for 7 days to IH (alternating 2 min 5% O2-15 min 21% O2), SH (5% O2) or RA (21% O2), then 3 glutamate challenges. The first and last exposures were intended as a metabolic stimulus (200 µM glutamate, 15 min); the second emulated excitotoxicity (10 mM glutamate, 10 min). GFAP, MAP2, and EAAT1, EAAT2 glutamate transporters expression were assessed after exposure to each hypoxic protocol. Additionally, cell viability was determined at baseline and after each glutamate challenge, in presence or absence of ceftriaxone that increases glutamate transporter expression. GFAP and MAP2 decreased after 7 days IH and SH. Long-term IH but not SH decreased EAAT1 and EAAT2. Excitotoxic glutamate challenge decreased cell viability and the following 200 µM exposure further increased cell death, particularly in IH-exposed slices. Ceftriaxone prevented glutamate transporter decrease and improved cell viability after IH and excitotoxicity. We conclude that IH is more detrimental to cell survival and glutamate homeostasis than SH. These findings suggest that impaired regulation of extracellular glutamate levels is implicated in the increased brain susceptibility to excitotoxic insult after long-term IH

Diabetic Microvascular Disease and Pulmonary Fibrosis: The Contribution of Platelets and Systemic Inflammation

Diabetes is strongly associated with systemic inflammation and oxidative stress, but its effect on pulmonary vascular disease and lung function has often been disregarded. Several studies identified restrictive lung disease and fibrotic changes in diabetic patients and in animal models of diabetes. While microvascular dysfunction is a well-known complication of diabetes, the mechanisms leading to diabetes-induced lung injury have largely been disregarded. We described the potential involvement of diabetes-induced platelet-endothelial interactions in perpetuating vascular inflammation and oxidative injury leading to fibrotic changes in the lung. Changes in nitric oxide synthase (NOS) activation and decreased NO bioavailability in the diabetic lung increase platelet activation and vascular injury and may account for platelet hyperreactivity reported in diabetic patients. Additionally, the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway has been reported to mediate pancreatic islet damage, and is implicated in the onset of diabetes, inflammation and vascular injury. Many growth factors and diabetes-induced agonists act via the JAK/STAT pathway. Other studies reported the contribution of the JAK/STAT pathway to the regulation of the pulmonary fibrotic process but the role of this pathway in the development of diabetic lung fibrosis has not been considered. These observations may open new therapeutic perspectives for modulating multiple pathways to mitigate diabetes onset or its pulmonary consequences

IH significantly decreases glutamate transporters, MAP2 and GFAP immunoreactivity.

<p>EAAT1, EAAT2, GFAP and MAP2 immunoreactivity in slices exposed to 7 days RA, SH or IH. EAAT1 and EAAT2 expression was unchanged by SH while significantly reduced in IH. MAP2 and GFAP expression decreased in both SH and IH.</p

Ceftriaxone effect on cell tolerance to glutamate is significantly greater in IH-exposed slices.

<p>Propidium iodide staining quantification of ceftriaxone (+) treated slices exposed to RA, SH and IH at baseline (BL), after 200 µM glutamate, and 10 mM glutamate, followed by a second 200 µM glutamate challenge. n = 12–18 for RA^-, SH^-, IH^- and n = 9-12 for RA+, SH⁺, IH⁺; IH^- > IH⁺: *At BL (p = .005), ^&At 10 mM (p<.001) & ∧At 200 µM#2 (p = .005).</p

Intermittent hypoxia decreases cell viability and impairs glutamate response.

<p>Propidium iodide staining (A) and quantification of PI positive cells presented as mean <u>+</u> SD (B) of slices exposed to 7 days RA, SH or IH at baseline (BL), after 200 µM glutamate, and 10 mM glutamate, followed by a second 200 µM glutamate challenge. n = 12–18. *: At all concentrations RA^- < SH^- & IH^- (p<.001). ⁺ At 10mM: SH^- < IH^- (p<.05). ^# At 10 mM & 200 µM#2: BL < SH^- & IH^- (p<.05 and p<.001 respectively).</p

Graphic representation of glutamate transporters, MAP2 and GFAP immunoreactivity with or without ceftriaxone.

<p>Quantification of EAAT1, EAAT2, GFAP and MAP2 immunofluorescence per unit area of slices exposed to 7 days RA, SH or IH in presence (+ve) or in absence (-ve) of 100 µM ceftriaxone. Data are presented as mean immunofluorescence <u>+</u> SD. n =  4-13 for RA⁺, IH⁺, SH⁺; n = 5–11 for RA^-, IH^-, SH *: IH^- or SH^- - (p≤0.01). ⁺: IH⁺ < SH⁺ (p≤0.01).</p

Long term sustained or Intermittent hypoxia decreases cell viability.

<p>Propidium iodide (red) and Fluorescein diacetate (green) staining of slices exposed to 7 days RA, SH or IH (A) without and (B) with 100 µM ceftriaxone (n = 4-6).</p