Sally H. Vitali

Exosomes Mediate the Cytoprotective Action of Mesenchymal Stromal Cells on Hypoxia-Induced Pulmonary Hypertension

Background— Hypoxia induces an inflammatory response in the lung manifested by alternative activation of macrophages with elevation of proinflammatory mediators that are critical for the later development of hypoxic pulmonary hypertension. Mesenchymal stromal cell transplantation inhibits lung inflammation, vascular remodeling, and right heart failure and reverses hypoxic pulmonary hypertension in experimental models of disease. In this study, we aimed to investigate the paracrine mechanisms by which mesenchymal stromal cells are protective in hypoxic pulmonary hypertension. Methods and Results— We fractionated mouse mesenchymal stromal cell–conditioned media to identify the biologically active component affecting in vivo hypoxic signaling and determined that exosomes, secreted membrane microvesicles, suppressed the hypoxic pulmonary influx of macrophages and the induction of proinflammatory and proproliferative mediators, including monocyte chemoattractant protein-1 and hypoxia-inducible mitogenic factor, in the murine model of hypoxic pulmonary hypertension. Intravenous delivery of mesenchymal stromal cell–derived exosomes (MEX) inhibited vascular remodeling and hypoxic pulmonary hypertension, whereas MEX-depleted media or fibroblast-derived exosomes had no effect. MEX suppressed the hypoxic activation of signal transducer and activator of transcription 3 (STAT3) and the upregulation of the miR-17 superfamily of microRNA clusters, whereas it increased lung levels of miR-204, a key microRNA, the expression of which is decreased in human pulmonary hypertension. MEX produced by human umbilical cord mesenchymal stromal cells inhibited STAT3 signaling in isolated human pulmonary artery endothelial cells, demonstrating a direct effect of MEX on hypoxic vascular cells. Conclusion— This study indicates that MEX exert a pleiotropic protective effect on the lung and inhibit pulmonary hypertension through suppression of hyperproliferative pathways, including STAT3-mediated signaling induced by hypoxia.

Mesenchymal stem cell-mediated reversal of bronchopulmonary dysplasia and associated pulmonary hypertension.

Clinical trials have failed to demonstrate an effective preventative or therapeutic strategy for bronchopulmonary dysplasia (BPD), a multifactorial chronic lung disease in preterm infants frequently complicated by pulmonary hypertension (PH). Mesenchymal stem cells (MSCs) and their secreted components have been shown to prevent BPD and pulmonary fibrosis in rodent models. We hypothesized that treatment with conditioned media (CM) from cultured mouse bone marrow-derived MSCs could reverse hyperoxia-induced BPD and PH. Newborn mice were exposed to hyperoxia (FiO2=0.75) for two weeks, were then treated with one intravenous dose of CM from either MSCs or primary mouse lung fibroblasts (MLFs), and placed in room air for two to four weeks. Histological analysis of lungs harvested at four weeks of age was performed to determine the degree of alveolar injury, blood vessel number, and vascular remodeling. At age six weeks, pulmonary artery pressure (PA acceleration time) and right ventricular hypertrophy (RVH; RV wall thickness) were assessed by echocardiography, and pulmonary function tests were conducted. When compared to MLF-CM, a single dose of MSC-CM-treatment (1) reversed the hyperoxia-induced parenchymal fibrosis and peripheral PA devascularization (pruning), (2) partially reversed alveolar injury, (3) normalized lung function (airway resistance, dynamic lung compliance), (4) fully reversed the moderate PH and RVH, and (5) attenuated peripheral PA muscularization associated with hyperoxia-induced BPD. Reversal of key features of hyperoxia-induced BPD and its long-term adverse effects on lung function can be achieved by a single intravenous dose of MSC-CM, thereby pointing toward a new therapeutic intervention for chronic lung diseases.

Genetic polymorphisms in sepsis

Context: Wide variability exists in the susceptibility to and outcome from sepsis even within similar intensive care unit populations. Some of this variability in the host may be due to genetic variation in genes coding for components of the innate immune response. Objective: To review the evidence for a genetic influence on the susceptibility to and outcome from sepsis. Design: Literature review. Patients: Variety of adult and pediatric patients with various critical illnesses and infections. Interventions: None. Main Outcome Measures: Susceptibility to clinical symptoms of sepsis and outcome as measured by severity of disease and mortality. Results: Polymorphisms in genes coding for proteins involved in the recognition of bacterial pathogens (Toll-like receptor 4, CD14, Fc&ggr;RIIa, and mannose-binding lectin) and the response to bacterial pathogens (tumor necrosis factor-&agr;, interleukin (IL)-1&agr;, IL-1&bgr;, IL-1 receptor agonist, IL-6, IL-10, heat shock proteins, angiotensin I converting enzyme, plasminogen activator inhibitor-1) can influence the amount or function of the protein produced in response to bacterial stimuli. Evidence is discussed suggesting that some of these genetic polymorphisms influence the susceptibility to and outcome from sepsis. Conclusion: Host genetic variability in the regulatory and coding regions of genes for components of the innate immune system may influence the susceptibility to and/or outcome from sepsis. The disparate results observed in many studies of polymorphisms in sepsis emphasize the need for future studies to be larger, to include the analysis of multiple polymorphisms, and to be better designed with respect to control populations to identify the degree of influence that genetic variability has on sepsis.

Mechanical ventilation guided by electrical impedance tomography in experimental acute lung injury.

Objective:To utilize real-time electrical impedance tomography to guide lung protective ventilation in an animal model of acute respiratory distress syndrome. Design:Prospective animal study. Setting:Animal research center. Subjects:Twelve Yorkshire swine (15 kg). Interventions:Lung injury was induced with saline lavage and augmented using large tidal volumes. The control group (n = 6) was ventilated using ARDSnet guidelines, and the electrical impedance tomography–guided group (n = 6) was ventilated using guidance with real-time electrical impedance tomography lung imaging. Regional electrical impedance tomography–derived compliance was used to maximize the recruitment of dependent lung and minimize overdistension of nondependent lung areas. Tidal volume was 6 mL/kg in both groups. Computed tomography was performed in a subset of animals to define the anatomic correlates of electrical impedance tomography imaging (n = 5). Interleukin-8 was quantified in serum and bronchoalveolar lavage samples. Sections of dependent and nondependent regions of the lung were fixed in formalin for histopathologic analysis. Measurements and Main Results:Positive end-expiratory pressure levels were higher in the electrical impedance tomography–guided group (14.3 cm H2O vs. 8.6 cm H2O; p < 0.0001), whereas plateau pressures did not differ. Global respiratory system compliance was improved in the electrical impedance tomography–guided group (6.9 mL/cm H2O vs. 4.7 mL/cm H2O; p = 0.013). Regional electrical impedance tomography–derived compliance of the most dependent lung region was increased in the electrical impedance tomography group (1.78 mL/cm H2O vs. 0.99 mL/cm H2O; p = 0.001). Pao2/FIO2 ratio was higher and oxygenation index was lower in the electrical impedance tomography–guided group (Pao2/FIO2: 388 mm Hg vs. 113 mm Hg, p < 0.0001; oxygentation index, 6.4 vs. 15.7; p = 0.02) (all averages over the 6-hr time course). The presence of hyaline membranes (HM) and airway fibrin (AF) was significantly reduced in the electrical impedance tomography–guided group (HMEIT 42% samples vs. HMCONTROL 67% samples, p < 0.01; AFEIT 75% samples vs. AFCONTROL 100% samples, p < 0.01). Interleukin-8 level (bronchoalveolar lavage) did not differ between the groups. The upper and lower 95% limits of agreement between electrical impedance tomography and computed tomography were ± 16%. Conclusions:Electrical impedance tomography–guided ventilation resulted in improved respiratory mechanics, improved gas exchange, and reduced histologic evidence of ventilator-induced lung injury in an animal model. This is the first prospective use of electrical impedance tomography–derived variables to improve outcomes in the setting of acute lung injury.

Impaired Vasoconstriction and Nitric Oxide-Mediated Relaxation in Pulmonary Arteries of Hypoxia- and Monocrotaline-Induced Pulmonary Hypertensive Rats

Pulmonary hypertension (PH) is a life-threatening disease with unclear vascular mechanisms. We tested whether PH involves abnormal pulmonary vasoconstriction and impaired vasodilation. Male Sprague-Dawley rats were exposed to hypoxia (9% O2) for 2 weeks or injected with single dose of monocrotaline (MCT, 60 mg/kg s.c.). Control rats were normoxic or injected with saline. After the hemodynamic measurements were performed, pulmonary and mesenteric arteries were isolated for measurement of vascular function. Hematocrit was elevated in hypoxic rats. Right ventricular systolic pressure and Fultons Index [right/(left + septum) ventricular weight] were greater in hypoxic and MCT-treated rats than in normoxic rats. Pulmonary artery contraction by phenylephrine and 96 mM KCl was less in hypoxic and MCT-treated rats than in normoxic rats. Acetylcholine-induced relaxation was less in the pulmonary arteries of hypoxic and MCT-treated rats than of normoxic rats, suggesting reduced effects of endothelium-derived vasodilators. The nitric oxide synthase inhibitor, Nω-nitro-l-arginine methyl ester, and the guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, inhibited acetylcholine relaxation, suggesting that it was mediated by nitric oxide (NO)-cGMP. The NO donor sodium nitroprusside caused less relaxation in the pulmonary arteries of hypoxic and MCT-treated than of normoxic rats, suggesting decreased responsiveness of vascular smooth muscle cells (VSMCs) to vasodilators. Phenylephrine and KCl contraction and acetylcholine and sodium nitroprusside relaxation were not different in the mesenteric arteries from all groups. In lung tissue sections, the wall thickness of pulmonary arterioles was greater in hypoxic and MCT-treated rats than in normoxic rats. The specific reductions in pulmonary, but not systemic, arterial vasoconstriction and vasodilation in hypoxia- and MCT-induced PH are consistent with the possibility of de-differentiation of pulmonary VSMCs to a more proliferative/synthetic and less contractile phenotype in PH.

Mesenchymal Stromal Cell Exosomes Ameliorate Experimental Bronchopulmonary Dysplasia and Restore Lung Function through Macrophage Immunomodulation

Rationale: Mesenchymal stem/stromal cell (MSC) therapies have shown promise in preclinical models of pathologies relevant to newborn medicine, such as bronchopulmonary dysplasia (BPD). We have reported that the therapeutic capacity of MSCs is comprised in their secretome, and demonstrated that the therapeutic vectors are exosomes produced by MSCs (MSC‐exos). Objectives: To assess efficacy of MSC‐exo treatment in a preclinical model of BPD and to investigate mechanisms underlying MSC‐exo therapeutic action. Methods: Exosomes were isolated from media conditioned by human MSC cultures. Newborn mice were exposed to hyperoxia (HYRX; 75% O2), treated with exosomes on Postnatal Day (PN) 4 and returned to room air on PN7. Treated animals and appropriate controls were harvested on PN7, ‐14, or ‐42 for assessment of pulmonary parameters. Measurements and Main Results: HYRX‐exposed mice presented with pronounced alveolar simplification, fibrosis, and pulmonary vascular remodeling, which was effectively ameliorated by MSC‐exo treatment. Pulmonary function tests and assessment of pulmonary hypertension showed functional improvements after MSC‐exo treatment. Lung mRNA sequencing demonstrated that MSC‐exo treatment induced pleiotropic effects on gene expression associated with HYRX‐induced inflammation and immune responses. MSC‐exos modulate the macrophage phenotype fulcrum, suppressing the proinflammatory “M1” state and augmenting an antiinflammatory “M2‐like” state, both in vitro and in vivo. Conclusions: MSC‐exo treatment blunts HYRX‐associated inflammation and alters the hyperoxic lung transcriptome. This results in alleviation of HYRX‐induced BPD, improvement of lung function, decrease in fibrosis and pulmonary vascular remodeling, and amelioration of pulmonary hypertension. The MSC‐exo mechanism of action is associated with modulation of lung macrophage phenotype.

The Sugen 5416/hypoxia mouse model of pulmonary hypertension revisited: long-term follow-up.

The combination of a vascular endothelial growth factor receptor antagonist, Sugen 5416 (SU5416), and chronic hypoxia is known to cause pronounced pulmonary hypertension (PH) with angioobliterative lesions in rats and leads to exaggerated PH in mice as well. We sought to determine whether weekly SU5416 injections during 3 weeks of hypoxia leads to long-term development of angioobliterative lesions and sustained or progressive PH in mice. Male C57BL/6J mice were injected with SU5416 (SuHx) or vehicle (VehHx) weekly during 3 weeks of exposure to 10% oxygen. Echocardiographic and invasive measures of hemodynamics and pulmonary vascular morphometry were performed after the 3-week hypoxic exposure and after 10 weeks of recovery in normoxia. SuHx led to higher right ventricular (RV) systolic pressure and RV hypertrophy than VehHx after 3 weeks of hypoxia. Ten weeks after hypoxic exposure, RV systolic pressure decreased but remained elevated in SuHx mice compared with VehHx or normoxic control mice, but RV hypertrophy had resolved. After 3 weeks of hypoxia and 10 weeks of follow-up in normoxia, tricuspid annular plane systolic excursion was significantly decreased, indicating decreased systolic RV function. Very few angioobliterative lesions were found at the 10-week follow-up time point in SuHx mouse lungs. In conclusion, SU5416 combined with 3 weeks of hypoxia causes a more profound PH phenotype in mice than hypoxia alone. PH persists over 10 weeks of normoxic follow-up in SuHx mice, but significant angioobliterative lesions do not occur, and neither PH nor RV dysfunction worsens. The SuHx mouse model is a useful adjunct to other PH models, but the search will continue for a mouse model that better recapitulates the human phenotype.

Assessing the quality of case-control association studies on the genetic basis of sepsis.

Objective: To define methodological standards that would improve the quality and reproducibility of case-control genetic association studies in sepsis. Design: Summary of the published standards. Results: Twelve main criteria and measures for evaluation of those criteria were developed from a variety of reviews and editorials on the subject. Conclusions: Case-control candidate gene association studies represent an important tool for understanding the role that genes play in the pathogenesis of sepsis. It is important, however, to understand the limitations of these studies and the need for replication.

Divergent cardiopulmonary actions of heme oxygenase enzymatic products in chronic hypoxia.

Background Hypoxia and pressure-overload induce heme oxygenase-1 (HO-1) in cardiomyocytes and vascular smooth muscle cells (VSMCs). HO-1−/− mice exposed to chronic hypoxia develop pulmonary arterial hypertension (PAH) with exaggerated right ventricular (RV) injury consisting of dilation, fibrosis, and mural thrombi. Our objective was to indentify the HO-1 product(s) mediating RV protection from hypoxic injury in HO-1−/− mice. Methodology/Principal Findings HO-1−/− mice were exposed to seven weeks of hypoxia and treated with inhaled CO or biliverdin injections. CO reduced right ventricular systolic pressure (RVSP) and prevented hypoxic pulmonary arteriolar remodeling in both HO-1−/− and control mice. Biliverdin had no significant effect on arteriolar remodeling or RVSP in either genotype. Despite this, biliverdin prevented RV failure in the hypoxic HO-1−/− mice (0/14 manifested RV wall fibrosis or thrombus), while CO-treated HO-1−/− mice developed RV insults similar to untreated controls. In vitro, CO inhibited hypoxic VSMC proliferation and migration but did not prevent cardiomyocyte death from anoxia-reoxygenation (A-R). In contrast, bilirubin limited A-R-induced cardiomyocyte death but did not inhibit VSMC proliferation and migration. Conclusions/Significance CO and bilirubin have distinct protective actions in the heart and pulmonary vasculature during chronic hypoxia. Moreover, reducing pulmonary vascular resistance may not prevent RV injury in hypoxia-induced PAH; supporting RV adaptation to hypoxia and preventing RV failure must be a therapeutic goal.

Continuous infusion, general anesthesia and other intensive care treatment for uncontrolled status epilepticus.

Purpose of review To discuss the use of continuous infusions, general anesthesia, hypothermia, and ketogenic diet as treatment for uncontrolled status epilepticus in pediatric patients. Recent findings Recent studies demonstrate that clinical practitioners have a hierarchy in approach in controlling refractory status epilepticus (RSE) and super-refractory status epilepticus in children. In the acute setting of RSE, midazolam achieves clinical seizure control at a mean of 41 min after starting an infusion. When midazolam has failed to control RSE, the evidence points to barbiturate anesthesia as the next frequently used option. When both midazolam and barbiturates have failed, use of isoflurane or ketamine anesthesia has been tried at a mean of 10 days after RSE onset, although the studies are largely anecdotal. Increasingly, the use of therapeutic hypothermia or ketogenic diet is described as a strategy for super-refractory status epilepticus, and better evidence for their use may become available from ongoing randomized studies. Summary Uncontrolled episodes of status epilepticus require intensive care treatment and the literature describes a common pathway of care used by many. However, cases of truly refractory and super-refractory status epilepticus are seen infrequently at any given institution. One strategy to improve the quality of evidence is to develop prospective, national and multinational case registries to determine the range of presentations and causes, efficacy of treatments, and clinical outcomes.