Skeletal Muscle SIRT3 Deficiency Contributes to Pulmonary Vascular Remodeling in Pulmonary Hypertension Due to Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Pulmonary hypertension (PH) is a major complication linked to adverse outcomes in heart failure with preserved ejection fraction (HFpEF), yet no specific therapies exist for PH associated with HFpEF (PH-HFpEF). We have recently reported on the role of skeletal muscle SIRT3 (sirtuin-3) in modulation of PH-HFpEF, suggesting a novel endocrine signaling pathway for skeletal muscle modulation of pulmonary vascular remodeling. In this study, we attempted to define the processes by which skeletal muscle SIRT3 defects affect pulmonary vascular health in PH-HFpEF. METHODS AND RESULTS: Skeletal muscle-specific Sirt3 knockout mice (Sirt3skm-/-) exhibited reduced pulmonary vascular density accompanied by pulmonary vascular proliferative remodeling and elevated pulmonary pressures. Using mass spectrometry-based comparative secretome analysis, we demonstrated elevated secretion of LOXL2 (lysyl oxidase homolog 2) in SIRT3-deficient skeletal muscle cells. Elevated circulation and protein expression levels of LOXL2 were also observed in plasma and skeletal muscle of Sirt3skm-/- mice, a rat model of PH-HFpEF, and humans with PH-HFpEF. In addition, expression levels of CNPY2 (canopy fibroblast growth factor signaling regulator 2), a known proliferative and angiogenic factor, were increased in pulmonary artery endothelial cells and pulmonary artery smooth muscle cells of Sirt3skm-/- mice and animal models of PH-HFpEF. CNPY2 levels were also higher in pulmonary artery smooth muscle cells of subjects with obesity compared with nonobese subjects. Moreover, treatment with recombinant LOXL2 protein promoted pulmonary artery endothelial cell migration/proliferation and pulmonary artery smooth muscle cell proliferation through regulation of CNPY2-p53 signaling. Last, skeletal muscle-specific Loxl2 deletion decreased pulmonary artery endothelial cell and pulmonary artery smooth muscle cell expression of CNPY2 and improved pulmonary pressures in mice with high-fat diet-induced PH-HFpEF. CONCLUSIONS: This study demonstrates a systemic pathogenic impact of skeletal muscle SIRT3 deficiency in remote pulmonary vascular remodeling and PH-HFpEF. This study suggests a new endocrine signaling axis that links skeletal muscle health and SIRT3 deficiency to remote CNPY2 regulation in the pulmonary vasculature through myokine LOXL2. Our data also identify skeletal muscle SIRT3, myokine LOXL2, and CNPY2 as potential targets for the treatment of PH-HFpEF.

Plasma Proteomics Identifies B2M as a Regulator of Pulmonary Hypertension in Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Pulmonary hypertension (PH) represents an important phenotype in heart failure with preserved ejection fraction (HFpEF). However, management of PH-HFpEF is challenging because mechanisms involved in the regulation of PH-HFpEF remain unclear. METHODS: We used a mass spectrometry-based comparative plasma proteomics approach as a sensitive and comprehensive hypothesis-generating discovery technique to profile proteins in patients with PH-HFpEF and control subjects. We then validated and investigated the role of one of the identified proteins using in vitro cell cultures, in vivo animal models, and independent cohort of human samples. RESULTS: Plasma proteomics identified high protein abundance levels of B2M (ß2-microglobulin) in patients with PH-HFpEF. Interestingly, both circulating and skeletal muscle levels of B2M were increased in mice with skeletal muscle SIRT3 (sirtuin-3) deficiency or high-fat diet-induced PH-HFpEF. Plasma and muscle biopsies from a validation cohort of PH-HFpEF patients were found to have increased B2M levels, which positively correlated with disease severity, especially pulmonary capillary wedge pressure and right atrial pressure at rest. Not only did the administration of exogenous B2M promote migration/proliferation in pulmonary arterial vascular endothelial cells but it also increased PCNA (proliferating cell nuclear antigen) expression and cell proliferation in pulmonary arterial vascular smooth muscle cells. Finally, B2m deletion improved glucose intolerance, reduced pulmonary vascular remodeling, lowered PH, and attenuated RV hypertrophy in mice with high-fat diet-induced PH-HFpEF. CONCLUSIONS: Patients with PH-HFpEF display higher circulating and skeletal muscle expression levels of B2M, the magnitude of which correlates with disease severity. Our findings also reveal a previously unknown pathogenic role of B2M in the regulation of pulmonary vascular proliferative remodeling and PH-HFpEF. These data suggest that circulating and skeletal muscle B2M can be promising targets for the management of PH-HFpEF.

Effect of estrogen receptor α on cardiopulmonary adaptation to chronic developmental hypoxia in a rat model.

Humans living at high-altitude (HA) have adapted to this environment by increasing pulmonary vascular and alveolar growth. RNA sequencing data from a novel murine model that mimics this phenotypical response to HA suggested estrogen signaling via estrogen receptor alpha (ERα) may be involved in this adaptation. We hypothesized ERα was a key mediator in the cardiopulmonary adaptation to chronic hypoxia and sought to delineate the mechanistic role ERα contributes to this process by exposing novel loss-of-function ERα mutant (ERαMut) rats to simulated HA. ERα mutant or wild-type (wt) rats were exposed to normoxia or hypoxia starting at conception and continued postnatally until 6 wk of age. Both wt and ERαMut animals born and raised in hypoxia exhibited lower body mass and higher hematocrits, total alveolar volumes (Va), diffusion capacities of carbon monoxide (DLCO), pulmonary arteriole (PA) wall thickness, and Fulton indices than normoxia animals. Right ventricle adaptation was maintained in the setting of hypoxia. Although no major physiologic differences were seen between wt and ERαMut animals at either exposure, ERαMut animals exhibited smaller mean linear intercepts (MLI) and increased PA total and lumen areas. Hypoxia exposure or ERα loss-of-function did not affect lung mRNA abundance of vascular endothelial growth factor, angiopoietin 2, or apelin. Sexual dimorphisms were noted in PA wall thickness and PA lumen area in ERαMut rats. In summary, in room air-exposed rats and rats with peri- and postnatal hypoxia exposure, ERα loss-of-function was associated with decreased alveolar size (primarily driven by hypoxic animals) and increased PA remodeling.NEW & NOTEWORTHY By exposing novel loss-of-function estrogen receptor alpha (Erα) mutant rats to a novel model of human high-altitude exposure, we demonstrate that ERα has subtle but inconsistent effects on endpoints relevant to cardiopulmonary adaptation to chronic hypoxia. Given that we observed some histologic, sex, and genotype differences, further research into cell-specific effects of ERα during hypoxia-induced cardiopulmonary adaptation is warranted.

SOX17 Deficiency Mediates Pulmonary Hypertension: At the Crossroads of Sex, Metabolism, and Genetics.

Rationale: Genetic studies suggest that SOX17 (SRY-related HMG-box 17) deficiency increases pulmonary arterial hypertension (PAH) risk. Objectives: On the basis of pathological roles of estrogen and HIF2α (hypoxia-inducible factor 2α) signaling in pulmonary artery endothelial cells (PAECs), we hypothesized that SOX17 is a target of estrogen signaling that promotes mitochondrial function and attenuates PAH development via HIF2α inhibition. Methods: We used metabolic (Seahorse) and promoter luciferase assays in PAECs together with the chronic hypoxia murine model to test the hypothesis. Measurements and Main Results: Sox17 expression was reduced in PAH tissues (rodent models and from patients). Chronic hypoxic pulmonary hypertension was exacerbated by mice with conditional Tie2-Sox17 (Sox17EC-/-) deletion and attenuated by transgenic Tie2-Sox17 overexpression (Sox17Tg). On the basis of untargeted proteomics, metabolism was the top pathway altered by SOX17 deficiency in PAECs. Mechanistically, we found that HIF2α concentrations were increased in the lungs of Sox17EC-/- and reduced in those from Sox17Tg mice. Increased SOX17 promoted oxidative phosphorylation and mitochondrial function in PAECs, which were partly attenuated by HIF2α overexpression. Rat lungs in males displayed higher Sox17 expression versus females, suggesting repression by estrogen signaling. Supporting 16α-hydroxyestrone (16αOHE; a pathologic estrogen metabolite)-mediated repression of SOX17 promoter activity, Sox17Tg mice attenuated 16αOHE-mediated exacerbations of chronic hypoxic pulmonary hypertension. Finally, in adjusted analyses in patients with PAH, we report novel associations between a SOX17 risk variant, rs10103692, and reduced plasma citrate concentrations (n = 1,326). Conclusions: Cumulatively, SOX17 promotes mitochondrial bioenergetics and attenuates PAH, in part, via inhibition of HIF2α. 16αOHE mediates PAH development via downregulation of SOX17, linking sexual dimorphism and SOX17 genetics in PAH.

Complex enameloid microstructure of †Ischyrhiza mira rostral denticles.

Serving in a foraging or self-defense capacity, pristiophorids, pristids, and the extinct sclerorhynchoids independently evolved an elongated rostrum lined with modified dermal denticles called rostral denticles. Isolated rostral denticles of the sclerorhynchoid Ischyrhiza mira are commonly recovered from Late Cretaceous North American marine deposits. Although the external morphology has been thoroughly presented in the literature, very little is known about the histological composition and organization of these curious structures. Using acid-etching techniques and scanning electron microscopy, we show that the microstructure of I. mira rostral denticles are considerably more complex than that of previously described dermal denticles situated elsewhere on the body. The apical cap consists of outer single crystallite enameloid (SCE) and inner bundled crystallite enameloid (BCE) overlying a region of orthodentine. The BCE has distinct parallel bundled enameloid (PBE), tangled bundled enameloid (TBE), and radial bundled enameloid (RBE) components. Additionally, the cutting edge of the rostral denticle is produced by a superficial layer of SCE and a deeper ridges/cutting edge layer (RCEL) of the BCE. The highly organized enameloid observed in the rostral denticles of this batomorph resembles that of the multifaceted tissue architecture observed in the oral teeth of selachimorphs and demonstrates that dermal scales have the capacity to evolve histologically similar complex tooth-like structures both inside and outside the oropharyngeal cavity.

17ß-Estradiol and estrogen receptor α protect right ventricular function in pulmonary hypertension via BMPR2 and apelin.

Women with pulmonary arterial hypertension (PAH) exhibit better right ventricular (RV) function and survival than men; however, the underlying mechanisms are unknown. We hypothesized that 17ß-estradiol (E2), through estrogen receptor α (ER-α), attenuates PAH-induced RV failure (RVF) by upregulating the procontractile and prosurvival peptide apelin via a BMPR2-dependent mechanism. We found that ER-α and apelin expression were decreased in RV homogenates from patients with RVF and from rats with maladaptive (but not adaptive) RV remodeling. RV cardiomyocyte apelin abundance increased in vivo or in vitro after treatment with E2 or ER-α agonist. Studies employing ER-α-null or ER-ß-null mice, ER-α loss-of-function mutant rats, or siRNA demonstrated that ER-α is necessary for E2 to upregulate RV apelin. E2 and ER-α increased BMPR2 in pulmonary hypertension RVs and in isolated RV cardiomyocytes, associated with ER-α binding to the Bmpr2 promoter. BMPR2 is required for E2-mediated increases in apelin abundance, and both BMPR2 and apelin are necessary for E2 to exert RV-protective effects. E2 or ER-α agonist rescued monocrotaline pulmonary hypertension and restored RV apelin and BMPR2. We identified what we believe to be a novel cardioprotective E2/ER-α/BMPR2/apelin axis in the RV. Harnessing this axis may lead to novel RV-targeted therapies for PAH patients of either sex.

Transcriptomic modifications in developmental cardiopulmonary adaptations to chronic hypoxia using a murine model of simulated high-altitude exposure.

Mechanisms driving adaptive developmental responses to chronic high-altitude (HA) exposure are incompletely known. We developed a novel rat model mimicking the human condition of cardiopulmonary adaptation to HA starting at conception and spanning the in utero and postnatal timeframe. We assessed lung growth and cardiopulmonary structure and function and performed transcriptome analyses to identify mechanisms facilitating developmental adaptations to chronic hypoxia. To generate the model, breeding pairs of Sprague-Dawley rats were exposed to hypobaric hypoxia (equivalent to 9,000 ft elevation). Mating, pregnancy, and delivery occurred in hypoxic conditions. Six weeks postpartum, structural and functional data were collected in the offspring. RNA-Seq was performed on right ventricle (RV) and lung tissue. Age-matched breeding pairs and offspring under room air (RA) conditions served as controls. Hypoxic rats exhibited significantly lower body weights and higher hematocrit levels, alveolar volumes, pulmonary diffusion capacities, RV mass, and RV systolic pressure, as well as increased pulmonary artery remodeling. RNA-Seq analyses revealed multiple differentially expressed genes in lungs and RVs from hypoxic rats. Although there was considerable similarity between hypoxic lungs and RVs compared with RA controls, several upstream regulators unique to lung or RV were identified. We noted a pattern of immune downregulation and regulation patterns of immune and hormonal mediators similar to the genome from patients with pulmonary arterial hypertension. In summary, we developed a novel murine model of chronic hypoxia exposure that demonstrates functional and structural phenotypes similar to human adaptation. We identified transcriptomic alterations that suggest potential mechanisms for adaptation to chronic HA.

Treatment With Treprostinil and Metformin Normalizes Hyperglycemia and Improves Cardiac Function in Pulmonary Hypertension Associated With Heart Failure With Preserved Ejection Fraction.

OBJECTIVE: Pulmonary hypertension (PH) due to left heart disease (group 2), especially in the setting of heart failure with preserved ejection fraction (HFpEF), is the most common cause of PH worldwide; however, at present, there is no proven effective therapy available for its treatment. PH-HFpEF is associated with insulin resistance and features of metabolic syndrome. The stable prostacyclin analog, treprostinil, is an effective and widely used Food and Drug Administration-approved drug for the treatment of pulmonary arterial hypertension. While the effect of treprostinil on metabolic syndrome is unknown, a recent study suggests that the prostacyclin analog beraprost can improve glucose intolerance and insulin sensitivity. We sought to evaluate the effectiveness of treprostinil in the treatment of metabolic syndrome-associated PH-HFpEF. Approach and Results: Treprostinil treatment was given to mice with mild metabolic syndrome-associated PH-HFpEF induced by high-fat diet and to SU5416/obese ZSF1 rats, a model created by the treatment of rats with a more profound metabolic syndrome due to double leptin receptor defect (obese ZSF1) with a vascular endothelial growth factor receptor blocker SU5416. In high-fat diet-exposed mice, chronic treatment with treprostinil reduced hyperglycemia and pulmonary hypertension. In SU5416/Obese ZSF1 rats, treprostinil improved hyperglycemia with similar efficacy to that of metformin (a first-line drug for type 2 diabetes mellitus); the glucose-lowering effect of treprostinil was further potentiated by the combined treatment with metformin. Early treatment with treprostinil in SU5416/Obese ZSF1 rats lowered pulmonary pressures, and a late treatment with treprostinil together with metformin improved pulmonary artery acceleration time to ejection time ratio and tricuspid annular plane systolic excursion with AMPK (AMP-activated protein kinase) activation in skeletal muscle and the right ventricle. CONCLUSIONS: Our data suggest a potential use of treprostinil as an early treatment for mild metabolic syndrome-associated PH-HFpEF and that combined treatment with treprostinil and metformin may improve hyperglycemia and cardiac function in a more severe disease.

Investigational new drug enabling angiotensin oral-delivery studies to attenuate pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a deadly and uncurable disease characterized by remodeling of the pulmonary vasculature and increased pulmonary artery pressure. Angiotensin Converting Enzyme 2 (ACE2) and its product, angiotensin-(1-7) [ANG-(1-7)] were expressed in lettuce chloroplasts to facilitate affordable oral drug delivery. Lyophilized lettuce cells were stable up to 28 months at ambient temperature with proper folding, assembly of CTB-ACE2/ANG-(1-7) and functionality. When the antibiotic resistance gene was removed, Ang1-7 expression was stable in subsequent generations in marker-free transplastomic lines. Oral gavage of monocrotaline-induced PAH rats resulted in dose-dependent delivery of ANG-(1-7) and ACE2 in plasma/tissues and PAH development was attenuated with decreases in right ventricular (RV) hypertrophy, RV systolic pressure, total pulmonary resistance and pulmonary artery remodeling. Such attenuation correlated well with alterations in the transcription of Ang-(1-7) receptor MAS and angiotensin II receptor AGTRI as well as IL-1ß and TGF-ß1. Toxicology studies showed that both male and female rats tolerated ~10-fold ACE2/ANG-(1-7) higher than efficacy dose. Plant cell wall degrading enzymes enhanced plasma levels of orally delivered protein drug bioencapsulated within plant cells. Efficient attenuation of PAH with no toxicity augurs well for clinical advancement of the first oral protein therapy to prevent/treat underlying pathology for this disease.

Therapeutic Use of Adipose-Derived Stromal Cells in a Murine Model of Acute Pancreatitis.

BACKGROUND: No specific therapy exists for acute pancreatitis (AP), and current treatment remains entirely supportive. Adipose stem cells (ASCs) have significant immunomodulatory and regenerative activities. We hypothesized that systemic administration of ASCs would mitigate inflammation in AP. METHODS: AP was induced in mice by 6 hourly intraperitoneal injections of cerulein. Twenty-four hours after AP induction, mice were randomized into four systemic treatment groups: sham group (no acute pancreatitis), vehicle, human ASCs, and human ASC-conditioned media. Mice were sacrificed at 48 h, and blood and organs were collected and analyzed. Pancreatic injury was quantified histologically using a published score (edema, inflammation, and necrosis). Pancreatic inflammation was also studied by immunohistochemistry and PCR. RESULTS: When using IV infusion of Hoechst-labeled ASCs, ASCs were found to localize to inflamed tissues: lungs and pancreas. Mice treated with ASCs had less severe AP, as shown by a significantly decreased histopathology score (edema, inflammation, and necrosis) (p = 0.001). ASCs infusion polarized pancreatic macrophages toward an anti-inflammatory M2 phenotype. ASC-conditioned media reduced pancreatic inflammation similarly to ASCs only, highlighting the importance of ASCs secreted factors in modulating inflammation. CONCLUSION: Intravenous delivery of human ASCs markedly reduces pancreatic inflammation in a murine model of AP ASCs which represent an effective therapy for AP.

Complementary Embryonic and Adult Cell Populations Enhance Myocardial Repair in Rat Myocardial Injury Model.

We compared the functional outcome of Isl-1+ cardiac progenitors, CD90+ bone marrow-derived progenitor cells, and the combination of the two in a rat myocardial infarction (MI) model. Isl-1+ cells were isolated from embryonic day 12.5 (E12.5) rat hearts and expanded in vitro. Thy-1+/CD90+ cells were isolated from the bone marrow of adult Sprague-Dawley rats by immunomagnetic cell sorting. Six-week-old female Sprague-Dawley rats underwent permanent left anterior descending (LAD) coronary artery ligation and received intramyocardial injection of either saline, Isl-1+ cells, CD90+ cells, or a combination of Isl-1+ and CD90+ cells, at the time of infarction. Cells were delivered transepicardially to the peri-infarct zone. Left ventricular function was assessed by transthoracic echocardiography at 1- and 4-week post-MI and by Millar catheterization (-dP/dt and +dP/dt) at 4-week post-MI. Fluorescence in situ hybridization (Isl-1+cells) and monochrystalline iron oxide nanoparticles labeling (MION; CD90+ cells) were performed to assess biodistribution of transplanted cells. Only the combination of cells demonstrated a significant improvement of cardiac function as assessed by anterior wall contractility, dP/dt (max), and dP/dt (min), compared to Isl-1+ or CD90+ cell monotherapies. In the combination cell group, viable cells were detected at week 4 when anterior wall motion was completely restored. In conclusion, the combination of Isl-1+ cardiac progenitors and adult bone marrow-derived CD90+ cells shows prolonged and robust myocardial tissue repair and provides support for the use of complementary cell populations to enhance myocardial repair.

Cigarette Smoking Impairs Adipose Stromal Cell Vasculogenic Activity and Abrogates Potency to Ameliorate Ischemia.

Cigarette smoking (CS) adversely affects the physiologic function of endothelial progenitor, hematopoietic stem and progenitor cells. However, the effect of CS on the ability of adipose stem/stromal cells (ASC) to promote vasculogenesis and rescue perfusion in the context of ischemia is unknown. To evaluate this, ASC from nonsmokers (nCS-ASC) and smokers (CS-ASC), and their activity to promote perfusion in hindlimb ischemia models, as well as endothelial cell (EC) survival and vascular morphogenesis in vitro were assessed. While nCS-ASC improved perfusion in ischemic limbs, CS-ASC completely lost this therapeutic effect. In vitro vasculogenesis assays revealed that human CS-ASC and ASC from CS-exposed mice showed compromised support of EC morphogenesis into vascular tubes, and the CS-ASC secretome was less potent in supporting EC survival/proliferation. Comparative secretome analysis revealed that CS-ASC produced lower amounts of hepatocyte growth factor (HGF) and stromal cell-derived growth factor 1 (SDF-1). Conversely, CS-ASC secreted the angiostatic/pro-inflammatory factor Activin A, which was not detected in nCS-ASC conditioned media (CM). Furthermore, higher Activin A levels were measured in EC/CS-ASC cocultures than in EC/nCS-ASC cocultures. CS-ASC also responded to inflammatory cytokines with 5.2-fold increase in Activin A secretion, whereas nCS-ASC showed minimal Activin A induction. Supplementation of EC/CS-ASC cocultures with nCS-ASC CM or with recombinant vascular endothelial growth factor, HGF, or SDF-1 did not rescue vasculogenesis, whereas inhibition of Activin A expression or activity improved network formation up to the level found in EC/nCS-ASC cocultures. In conclusion, ASC of CS individuals manifest compromised in vitro vasculogenic activity as well as in vivo therapeutic activity. Stem Cells 2018;36:856-867.

Mucosal Perfusion Preservation by a Novel Shapeable Tissue Expander for Oral Reconstruction.

BACKGROUND: There are few methods for expanding oral mucosa, and these often cause complications such as tissue necrosis and expander eruption. This study examines mucosal blood perfusion following insertion of a novel shapeable hydrogel tissue expander (HTE). The canine model used subgingival insertion of HTE following tooth extraction and alveolar bone reduction. The primary goal of this study was to gain understanding of epithelial perfusion and reparative responses of gingival mucosa during HTE expansion. METHODS: Nine Beagle dogs underwent bilateral premolar maxillary and mandibular tooth extraction. Three to four months later, HTE-contoured inserts were implanted submucosally under the buccal surface of the alveolar ridge. After removal and following a 6- to 7-month period of healing, new HTE implants were inserted at the same sites. The area was assessed weekly for tissue perfusion and volume of expansion. Biopsies for histological analysis were performed at the time of expander removal. RESULTS: Within 2 weeks following the second insertion, blood flow returned to baseline (defined as the values of perfusion measurements at the presurgery assessment) and remained normal until hydrogel full expansion and removal. Volume expansion analysis revealed that the hydrogel doubled in volume. Histological assessment showed no macrophage or inflammatory infiltration of the mucosa. No superficial fibrosis, decreased vascularity, or mucosal change was seen. CONCLUSION: Maintenance of adequate tissue perfusion is a clinically important aspect of tissue expander performance to reduce risk of device loss or injury to the patient, particularly for areas with a history of previous surgeries.

Human Adipose-Derived Stem Cells Suppress Elastase-Induced Murine Abdominal Aortic Inflammation and Aneurysm Expansion Through Paracrine Factors.

Abdominal aortic aneurysm (AAA) is a potentially lethal disease associated with immune activation-induced aortic degradation. We hypothesized that xenotransplantation of human adipose-derived stem cells (hADSCs) would reduce aortic inflammation and attenuate expansion in a murine AAA model. Modulatory effects of ADSCs on immune cell subtypes associated with AAA progression were investigated using human peripheral blood mononuclear cells (hPBMNCs) cocultured with ADSCs. Murine AAA was induced through elastase application to the abdominal aorta in C57BL/6 mice. ADSCs were administered intravenously, and aortic changes were determined by ultrasonography and videomicrometry. Circulating monocytes, aortic neutrophils, CD28- T cells, FoxP3+ regulatory T cells (Tregs), and CD206+ M2 macrophages were assessed at multiple terminal time points. In vitro, ADSCs induced M2 macrophage and Treg phenotypes while inhibiting neutrophil transmigration and lymphocyte activation without cellular contact. Intravenous ADSC delivery reduced aneurysmal expansion starting from day 4 [from baseline: 54.8% (saline) vs. 16.9% (ADSCs), n = 10 at baseline, n = 4 at day 4, p < 0.001], and the therapeutic effect persists through day 14 (from baseline: 64.1% saline vs. 24.6% ADSCs, n = 4, p < 0.01). ADSC administration increased aortic Tregs by 20-fold (n = 5, p < 0.01), while decreasing CD4+CD28- (-28%), CD8+CD28- T cells (-61%), and Ly6G/C+ neutrophils (-43%, n = 5, p < 0.05). Circulating CD115+CXCR1-LY6C+-activated monocytes decreased in the ADSC-treated group by day 7 (-60%, n = 10, p < 0.05), paralleled by an increase in aortic CD206+ M2 macrophages by 2.4-fold (n = 5, p < 0.05). Intravenously injected ADSCs transiently engrafted in the lung on day 1 without aortic engraftment at any time point. In conclusion, ADSCs exhibit pleiotropic immunomodulatory effects in vitro as well as in vivo during the development of AAA. The temporal evolution of these effects systemically as well as in aortic tissue suggests that ADSCs induce a sequence of anti-inflammatory cellular events mediated by paracrine factors, which leads to amelioration of AAA progression.

17ß-Estradiol mediates superior adaptation of right ventricular function to acute strenuous exercise in female rats with severe pulmonary hypertension.

17ß-Estradiol (E2) exerts protective effects on right ventricular (RV) function in pulmonary arterial hypertension (PAH). Since acute exercise-induced increases in afterload may lead to RV dysfunction in PAH, we sought to determine whether E2 allows for superior RV adaptation after an acute exercise challenge. We studied echocardiographic, hemodynamic, structural, and biochemical markers of RV function in male and female rats with sugen/hypoxia (SuHx)-induced pulmonary hypertension, as well as in ovariectomized (OVX) SuHx females, with or without concomitant E2 repletion (75 µg·kg(-1)·day(-1)) immediately after 45 min of treadmill running at 75% of individually determined maximal aerobic capacity (75% aerobic capacity reserve). Compared with males, intact female rats exhibited higher stroke volume and cardiac indexes, a strong trend for better RV compliance, and less pronounced increases in indexed total pulmonary resistance. OVX abrogated favorable RV adaptations, whereas E2 repletion after OVX markedly improved RV function. E2's effects on pulmonary vascular remodeling were complex and less robust than its RV effects. Postexercise hemodynamics in females with endogenous or exogenous E2 were similar to hemodynamics in nonexercised controls, whereas OVX rats exhibited more severely altered postexercise hemodynamics. E2 mediated inhibitory effects on RV fibrosis and attenuated increases in RV collagen I/III ratio. Proapoptotic signaling, endothelial nitric oxide synthase phosphorylation, and autophagic flux markers were affected by E2 depletion and/or repletion. Markers of impaired autophagic flux correlated with endpoints of RV structure and function. Endogenous and exogenous E2 exerts protective effects on RV function measured immediately after an acute exercise challenge. Harnessing E2's mechanisms may lead to novel RV-directed therapies.

Pulmonary Retention of Adipose Stromal Cells Following Intravenous Delivery Is Markedly Altered in the Presence of ARDS.

Transplantation of mesenchymal stromal cells (MSCs) has been shown to effectively prevent lung injury in several preclinical models of acute respiratory distress syndrome (ARDS). Since MSC therapy is tested in clinical trials for ARDS, there is an increased need to define the dynamics of cell trafficking and organ-specific accumulation. We examined how the presence of ARDS changes retention and organ-specific distribution of intravenously delivered MSCs isolated from subcutaneous adipose tissue [adipose-derived stem cells (ADSCs)]. This type of cell therapy was previously shown to ameliorate ARDS pathology. ARDS was triggered by lipopolysaccharide (LPS) aspiration, 4 h after which 300,000 murine CRE+ ADSCs were delivered intravenously. The distribution of ADSCs in the lungs and other organs was assessed by real-time polymerase chain reaction (PCR) of genomic DNA. As anticipated, the majority of delivered ADSCs accumulated in the lungs of both control and LPS-challenged mice, with minor amounts distributed to the liver, kidney, spleen, heart, and brain. Interestingly, within 2 h following ADSC administration, LPS-challenged lungs retained significantly lower levels of ADSCs compared to control lungs (∼7% vs. 15% of the original dose, respectively), whereas the liver, kidney, spleen, and brain of ARDS-affected animals retained significantly higher numbers of ADSCs compared to control animals. In contrast, 48 h later, only LPS-challenged lungs continued to retain ADSCs (∼3% of the original dose), whereas the lungs of control animals and nonpulmonary organs in either control or ARDS mice had no detectable levels of ADSCs. Our data suggest that the pulmonary microenvironment during ARDS may lessen the pulmonary capillary occlusion by MSCs immediately following cell delivery while facilitating pulmonary retention of the cells.

Conditioned media from adipose stromal cells limit lipopolysaccharide-induced lung injury, endothelial hyperpermeability and apoptosis.

BACKGROUND: Acute Respiratory Distress Syndrome (ARDS) is a condition that contributes to morbidity and mortality of critically ill patients. We investigated whether factors secreted by adipose stromal cells (ASC) into conditioned media (ASC-CM) will effectively decrease lung injury in the model of lipopolysaccharide (LPS)-induced ARDS. METHODS: To assess the effect of ASC-CM on ARDS indices, intravenous delivery of ASC and ASC-CM to C57Bl/6 mice was carried out 4 h after LPS oropharyngeal aspiration; Evans Blue Dye (EBD) was injected intravenously 1 h prior to animal sacrifice (48 h post-LPS). Lungs were either fixed for histopathology, or used to extract bronchoalveolar lavage fluid (BALF) or EBD. To assess the effect of ASC-CM on endothelial barrier function and apoptosis, human pulmonary artery endothelial cells were treated with ASC-CM for 48-72 h. RESULTS: ASC-CM markedly reduced LPS-induced histopathologic changes of lung, protein extravasation into BALF, and suppressed the secretion of proinflammatory cytokines TNFα and IL6. White Blood Cells (WBC) from BALF of LPS-challenged mice receiving ASC-CM had decreased reactive oxygen species (ROS) generation compared to WBC from LPS-challenged mice receiving control media injection. Treatment of pulmonary endothelial monolayers with ASC-CM significantly suppressed H2O2-induced leakage of FITC dextran and changes in transendothelial resistance, as well as gap formation in endothelial monolayer. ASC-CM exposure reduced the percentage of endothelial cells expressing ICAM-1, and suppressed TNFα-induced expression of E-selectin and cleavage of caspase-3. ASC-CM reduced the endothelial level of pro-apoptotic protein Bim, but did not affect the level of Bcl-2, Bad, or Bad phosphorylation. CONCLUSIONS: Factors secreted by ASC efficiently reduce ARDS indices, endothelial barrier hyperpermeability, and activation of pro-inflammatory and pro-apoptotic pathways in endothelium.

Human adipose-derived stem cells ameliorate cigarette smoke-induced murine myelosuppression via secretion of TSG-6.

OBJECTIVE: Bone marrow-derived hematopoietic stem and progenitor cells (HSC/HPC) are critical to homeostasis and tissue repair. The aims of this study were to delineate the myelotoxicity of cigarette smoking (CS) in a murine model, to explore human adipose-derived stem cells (hASC) as a novel approach to mitigate this toxicity, and to identify key mediating factors for ASC activities. METHODS: C57BL/6 mice were exposed to CS with or without i.v. injection of regular or siRNA-transfected hASC. For in vitro experiments, cigarette smoke extract was used to mimic the toxicity of CS exposure. Analysis of bone marrow HPC was performed both by flow cytometry and colony-forming unit assays. RESULTS: In this study, we demonstrate that as few as 3 days of CS exposure results in marked cycling arrest and diminished clonogenic capacity of HPC, followed by depletion of phenotypically defined HSC/HPC. Intravenous injection of hASC substantially ameliorated both acute and chronic CS-induced myelosuppression. This effect was specifically dependent on the anti-inflammatory factor TSG-6, which is induced from xenografted hASC, primarily located in the lung and capable of responding to host inflammatory signals. Gene expression analysis within bone marrow HSC/HPC revealed several specific signaling molecules altered by CS and normalized by hASC. CONCLUSION: Our results suggest that systemic administration of hASC or TSG-6 may be novel approaches to reverse CS-induced myelosuppression.

Muscle-derived Gr1(dim)CD11b(+) cells enhance neovascularization in an ischemic hind limb mouse model.

Gr1(+)CD11b(+) cells are characterized as myeloid-derived suppressor cells potentially involved in angiogenesis. We demonstrate that Gr1(+)CD11b(+) cells isolated from ischemic muscle in a hind-limb ischemic C57BL/6 mouse model play a role in vessel formation after ischemic injury. Gr1(dim)CD11b(+) cells, a subpopulation of Gr1(+)CD11b(+) cells, within skeletal muscle were increased in context of ischemia. Strikingly, astrocyte-plexus formed from muscle-derived Gr1(dim)CD11b(+) cells in Matrigel culture, followed by formation of isolectin and von Willebrand Factor-expressing cells, similar to that reported for angiogenesis in retina. When isolated muscle-derived Gr1(dim)CD11b(+) cells were injected into ischemic muscles, recovery of blood flow was significantly enhanced and these cells were incorporated into vessel walls. This suggests that Gr1(dim)CD11b(+) cells are recruited into ischemic regions after ischemia and may be involved in angiogenesis by their capacity to generate vascular cells.