LRRC15 inhibits SARS-CoV-2 cellular entry in trans.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is mediated by the entry receptor angiotensin-converting enzyme 2 (ACE2). Although attachment factors and coreceptors facilitating entry are extensively studied, cellular entry factors inhibiting viral entry are largely unknown. Using a surfaceome CRISPR activation screen, we identified human LRRC15 as an inhibitory attachment factor for SARS-CoV-2 entry. LRRC15 directly binds to the receptor-binding domain (RBD) of spike protein with a moderate affinity and inhibits spike-mediated entry. Analysis of human lung single-cell RNA sequencing dataset reveals that expression of LRRC15 is primarily detected in fibroblasts and particularly enriched in pathological fibroblasts in COVID-19 patients. ACE2 and LRRC15 are not coexpressed in the same cell types in the lung. Strikingly, expression of LRRC15 in ACE2-negative cells blocks spike-mediated viral entry in ACE2+ cell in trans, suggesting a protective role of LRRC15 in a physiological context. Therefore, LRRC15 represents an inhibitory attachment factor for SARS-CoV-2 that regulates viral entry in trans.

SOX17 Enhancer Variants Disrupt Transcription Factor Binding And Enhancer Inactivity Drives Pulmonary Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare disease characterized by remodeling of the pulmonary arteries, increased vascular resistance, and right-sided heart failure. Genome-wide association studies of idiopathic/heritable PAH established novel genetic risk variants, including conserved enhancers upstream of transcription factor (TF) SOX17 containing 2 independent signals. SOX17 is an important TF in embryonic development and in the homeostasis of pulmonary artery endothelial cells (hPAEC) in the adult. Rare pathogenic mutations in SOX17 cause heritable PAH. We hypothesized that PAH risk alleles in an enhancer region impair TF-binding upstream of SOX17, which in turn reduces SOX17 expression and contributes to disturbed endothelial cell function and PAH development. METHODS: CRISPR manipulation and siRNA were used to modulate SOX17 expression. Electromobility shift assays were used to confirm in silico-predicted TF differential binding to the SOX17 variants. Functional assays in hPAECs were used to establish the biological consequences of SOX17 loss. In silico analysis with the connectivity map was used to predict compounds that rescue disturbed SOX17 signaling. Mice with deletion of the SOX17-signal 1 enhancer region (SOX17-4593/enhKO) were phenotyped in response to chronic hypoxia and SU5416/hypoxia. RESULTS: CRISPR inhibition of SOX17-signal 2 and deletion of SOX17-signal 1 specifically decreased SOX17 expression. Electromobility shift assays demonstrated differential binding of hPAEC nuclear proteins to the risk and nonrisk alleles from both SOX17 signals. Candidate TFs HOXA5 and ROR-α were identified through in silico analysis and antibody electromobility shift assays. Analysis of the hPAEC transcriptomes revealed alteration of PAH-relevant pathways on SOX17 silencing, including extracellular matrix regulation. SOX17 silencing in hPAECs resulted in increased apoptosis, proliferation, and disturbance of barrier function. With the use of the connectivity map, compounds were identified that reversed the SOX17-dysfunction transcriptomic signatures in hPAECs. SOX17 enhancer knockout in mice reduced lung SOX17 expression, resulting in more severe pulmonary vascular leak and hypoxia or SU5416/hypoxia-induced pulmonary hypertension. CONCLUSIONS: Common PAH risk variants upstream of the SOX17 promoter reduce endothelial SOX17 expression, at least in part, through differential binding of HOXA5 and ROR-α. Reduced SOX17 expression results in disturbed hPAEC function and PAH. Existing drug compounds can reverse the disturbed SOX17 pulmonary endothelial transcriptomic signature.

Inhibition of lipid phosphatase SHIP1 expands myeloid-derived suppressor cells and attenuates rheumatoid arthritis in mice.

Rheumatoid arthritis (RA) is a debilitating autoimmune disease of unknown cause, characterized by infiltration and accumulation of activated immune cells in the synovial joints where cartilage and bone destructions occur. Myeloid-derived suppressor cells (MDSCs) are of myeloid origin and are able to suppress T cell responses. Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1) was shown to be involved in the regulation of MDSC differentiation. The purpose of the present study was to investigate the effect of inhibition of SHIP1 on the expansion of MDSCs in RA using a collagen-induced inflammatory arthritis (CIA) mouse model. In DBA/1 mice, treatment with a small molecule-specific SHIP1 inhibitor 3α-aminocholestane (3AC) induced a marked expansion of MDSCs in vivo. Both pretreatment with 3AC of DBA/1 mice prior to CIA induction and intervention with 3AC during CIA progression significantly reduced disease incidence and severity. Adoptive transfer of MDSCs isolated from 3AC-treated mice, but not naïve MDSCs from normal mice, into CIA mice significantly reduced disease incidence and severity, indicating that the 3AC-induced MDSCs were the cellular mediators of the observed amelioration of the disease. In conclusion, inhibition of SHIP1 expands MDSCs in vivo and attenuates development of CIA in mice. Small molecule-specific inhibition of SHIP1 may therefore offer therapeutic benefit to patients with RA and other autoimmune diseases.

Mesenchymal Stem Cell Extracellular Vesicles Reverse Sugen/Hypoxia Pulmonary Hypertension in Rats.

Mesenchymal stem cell extracellular vesicles attenuate pulmonary hypertension, but their ability to reverse established disease in larger animal models and the duration and mechanism(s) of their effect are unknown. We sought to determine the efficacy and mechanism of mesenchymal stem cells' extracellular vesicles in attenuating pulmonary hypertension in rats with Sugen/hypoxia-induced pulmonary hypertension. Male rats were treated with mesenchymal stem cell extracellular vesicles or an equal volume of saline vehicle by tail vein injection before or after subcutaneous injection of Sugen 5416 and exposure to 3 weeks of hypoxia. Pulmonary hypertension was assessed by right ventricular systolic pressure, right ventricular weight to left ventricle + septum weight, and muscularization of peripheral pulmonary vessels. Immunohistochemistry was used to measure macrophage activation state and recruitment to lung. Mesenchymal stem cell extracellular vesicles injected before or after induction of pulmonary hypertension normalized right ventricular pressure and reduced right ventricular hypertrophy and muscularization of peripheral pulmonary vessels. The effect was consistent over a range of doses and dosing intervals and was associated with lower numbers of lung macrophages, a higher ratio of alternatively to classically activated macrophages (M2/M1 = 2.00 ± 0.14 vs. 1.09 ± 0.11; P < 0.01), and increased numbers of peripheral blood vessels (11.8 ± 0.66 vs. 6.9 ± 0.57 vessels per field; P < 0.001). Mesenchymal stem cell extracellular vesicles are effective at preventing and reversing pulmonary hypertension in Sugen/hypoxia pulmonary hypertension and may offer a new approach for the treatment of pulmonary arterial hypertension.

Lipid phosphatase SHIP-1 regulates chondrocyte hypertrophy and skeletal development.

SH2-containing inositol-5'-phosphatase-1 (SHIP-1) controls the phosphatidylinositol-3'-kinase (PI3K) initiated signaling pathway by limiting cell membrane recruitment and activation of Akt. Despite the fact that many of the growth factors important to cartilage development and functions are able to activate the PI3K signal transduction pathway, little is known about the role of PI3K signaling in chondrocyte biology and its contribution to mammalian skeletogenesis. Here, we report that the lipid phosphatase SHIP-1 regulates chondrocyte hypertrophy and skeletal development through its expression in osteochondroprogenitor cells. Global SHIP-1 knockout led to accelerated chondrocyte hypertrophy and premature formation of the secondary ossification center in the bones of postnatal mice. Drastically higher vascularization and greater number of c-kit + progenitors associated with sinusoids in the bone marrow also indicated more advanced chondrocyte hypertrophic differentiation in SHIP-1 knockout mice than in wild-type mice. In corroboration with the in vivo phenotype, SHIP-1 deficient PDGFRα + Sca-1 + osteochondroprogenitor cells exhibited rapid differentiation into hypertrophic chondrocytes under chondrogenic culture conditions in vitro. Furthermore, SHIP-1 deficiency inhibited hypoxia-induced cellular activation of Akt and extracellular-signal-regulated kinase (Erk) and suppressed hypoxia-induced cell proliferation. These results suggest that SHIP-1 is required for hypoxia-induced growth signaling under physiological hypoxia in the bone marrow. In conclusion, the lipid phosphatase SHIP-1 regulates skeletal development by modulating chondrogenesis and the hypoxia response of the osteochondroprogenitors during endochondral bone formation.

Low dose 100 cGy irradiation as a potential therapy for pulmonary hypertension.

Pulmonary hypertension (PH) is an incurable disease characterized by pulmonary vascular remodeling and ultimately death. Two rodent models of PH include treatment with monocrotaline or exposure to a vascular endothelial growth factor receptor inhibitor and hypoxia. Studies in these models indicated that damaged lung cells evolve extracellular vesicles which induce production of progenitors that travel back to the lung and induce PH. A study in patients with pulmonary myelofibrosis and PH indicated that 100 cGy lung irradiation could remit both diseases. Previous studies indicated that murine progenitors were radiosensitive at very low doses, suggesting that 100 cGy treatment of mice with induced PH might be an effective PH therapy. Our hypothesis is that the elimination of the PH-inducing marrow cells by low dose irradiation would remove the cellular influences creating PH. Here we show that low dose whole-body irradiation can both prevent and reverse established PH in both rodent models of PH.

M1 Macrophage-Induced Endothelial-to-Mesenchymal Transition Promotes Infantile Hemangioma Regression.

Infantile hemangiomas are benign tumors of vascular endothelial cells (ECs), characterized by three distinct stages: proliferating phase, involuting phase, and involuted phase. The mechanisms that trigger involution of hemangioma into fibro-fatty tissue remain unknown. We report a novel mechanism by which M1-polarized macrophages induce endothelial-to-mesenchymal transition (EndMT) and promote hemangioma regression. M1- but not M2-polarized macrophages induced EndMT in ECs. Tumor necrosis factor-α and, to a lesser extent, IL-1ß and interferon-γ were the most potent cytokines produced by the M1 macrophages that induce in vitro EndMT. Western blot analysis and gene expression profiling showed that ECs treated with M1 macrophages, tumor necrosis factor-α, or IL-1ß decreased the expression of endothelial markers, whereas mesenchymal markers increased concomitantly. Immunohistochemical staining of patient samples revealed that a significant perivascular infiltration of M1, but not M2, macrophages coincides with endothelial expression of the critical EndMT transcription factors Snail/Slug in involuting hemangiomas. Most strikingly, M1 macrophage-treated ECs isolated from patient hemangiomas (HemECs) but not untreated HemECs readily differentiated into adipocytes on adipogenic induction. Thus, in vitro EndMT and adipogenesis of HemECs have, in part, recapitulated the natural history of hemangioma regression. In conclusion, our findings indicate that EndMT induced by M1 macrophages promotes infantile hemangioma regression and may lead to novel therapeutic treatments for this vascular tumor.

Light at the ENDothelium-role of Sox17 and Runx1 in endothelial dysfunction and pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease that is characterized by an obliterative vasculopathy of the distal pulmonary circulation. Despite significant progress in our understanding of the pathophysiology, currently approved medical therapies for PAH act primarily as pulmonary vasodilators and fail to address the underlying processes that lead to the development and progression of the disease. Endothelial dysregulation in response to stress, injury or physiologic stimuli followed by perivascular infiltration of immune cells plays a prominent role in the pulmonary vascular remodeling of PAH. Over the last few decades, our understanding of endothelial cell dysregulation has evolved and brought to light a number of transcription factors that play important roles in vascular homeostasis and angiogenesis. In this review, we examine two such factors, SOX17 and one of its downstream targets, RUNX1 and the emerging data that implicate their roles in the pathogenesis of PAH. We review their discovery and discuss their function in angiogenesis and lung vascular development including their roles in endothelial to hematopoietic transition (EHT) and their ability to drive progenitor stem cells toward an endothelial or myeloid fate. We also summarize the data from studies that link mutations in Sox17 with an increased risk of developing PAH and studies that implicate Sox17 and Runx1 in the pathogenesis of PAH. Finally, we review the results of recent studies from our lab demonstrating the efficacy of preventing and reversing pulmonary hypertension in animal models of PAH by deleting RUNX1 expression in endothelial or myeloid cells or by the use of RUNX1 inhibitors. By investigating PAH through the lens of SOX17 and RUNX1 we hope to shed light on the role of these transcription factors in vascular homeostasis and endothelial dysregulation, their contribution to pulmonary vascular remodeling in PAH, and their potential as novel therapeutic targets for treating this devastating disease.

Early macrophage recruitment and alternative activation are critical for the later development of hypoxia-induced pulmonary hypertension.

BACKGROUND: Lung inflammation precedes the development of hypoxia-induced pulmonary hypertension (HPH); however, its role in the pathogenesis of HPH is poorly understood. We sought to characterize the hypoxic inflammatory response and to elucidate its role in the development of HPH. We also aimed to investigate the mechanisms by which heme oxygenase-1, an anti-inflammatory enzyme, is protective in HPH. METHODS AND RESULTS: We generated bitransgenic mice that overexpress human heme oxygenase-1 under doxycycline control in an inducible, lung-specific manner. Hypoxic exposure of mice in the absence of doxycycline resulted in early transient accumulation of monocytes/macrophages in the bronchoalveolar lavage. Alveolar macrophages acquired an alternatively activated phenotype (M2) in response to hypoxia, characterized by the expression of found in inflammatory zone-1, arginase-1, and chitinase-3-like-3. A brief 2-day pulse of doxycycline delayed, but did not prevent, the peak of hypoxic inflammation, and could not protect against HPH. In contrast, a 7-day doxycycline treatment sustained high heme oxygenase-1 levels during the entire period of hypoxic inflammation, inhibited macrophage accumulation and activation, induced macrophage interleukin-10 expression, and prevented the development of HPH. Supernatants from hypoxic M2 macrophages promoted the proliferation of pulmonary artery smooth muscle cells, whereas treatment with carbon monoxide, a heme oxygenase-1 enzymatic product, abrogated this effect. CONCLUSIONS: Early recruitment and alternative activation of macrophages in hypoxic lungs are critical for the later development of HPH. Heme oxygenase-1 may confer protection from HPH by effectively modifying the macrophage activation state in hypoxia.

Mesenchymal stromal cells expressing heme oxygenase-1 reverse pulmonary hypertension.

Pulmonary arterial hypertension (PAH) remains a serious disease, and although current treatments may prolong and improve quality of life, search for novel and effective therapies is warranted. Using genetically modified mouse lines, we tested the ability of bone marrow-derived stromal cells (mesenchymal stem cells [MSCs]) to treat chronic hypoxia-induced PAH. Recipient mice were exposed for 5 weeks to normobaric hypoxia (8%-10% O(2)), MSC preparations were delivered through jugular vein injection and their effect on PAH was assessed after two additional weeks in hypoxia. Donor MSCs derived from wild-type (WT) mice or heme oxygenase-1 (HO-1) null mice (Hmox1(KO)) conferred partial protection from PAH when transplanted into WT or Hmox1(KO) recipients, whereas treatment with MSCs isolated from transgenic mice harboring a human HO-1 transgene under the control of surfactant protein C promoter (SH01 line) reversed established disease in WT recipients. SH01-MSC treatment of Hmox1(KO) animals, which develop right ventricular (RV) infarction under prolonged hypoxia, resulted in normal RV systolic pressure, significant reduction of RV hypertrophy and prevention of RV infarction. Donor MSCs isolated from a bitransgenic mouse line with doxycycline-inducible, lung-specific expression of HO-1 exhibited similar therapeutic efficacy only on doxycycline treatment of the recipients. In vitro experiments indicate that potential mechanisms of MSC action include modulation of hypoxia-induced lung inflammation and inhibition of smooth muscle cell proliferation. Cumulatively, our results demonstrate that MSCs ameliorate chronic hypoxia-induced PAH and their efficacy is highly augmented by lung-specific HO-1 expression in the transplanted cells, suggesting an interplay between HO-1-dependent and HO-1-independent protective pathways.

Targeting RUNX1 as a novel treatment modality for pulmonary arterial hypertension.

AIMS: Pulmonary arterial hypertension (PAH) is a fatal disease without a cure. Previously, we found that transcription factor RUNX1-dependent haematopoietic transformation of endothelial progenitor cells may contribute to the pathogenesis of PAH. However, the therapeutic potential of RUNX1 inhibition to reverse established PAH remains unknown. In the current study, we aimed to determine whether RUNX1 inhibition was sufficient to reverse Sugen/hypoxia (SuHx)-induced pulmonary hypertension (PH) in rats. We also aimed to demonstrate possible mechanisms involved. METHODS AND RESULTS: We administered a small molecule specific RUNX1 inhibitor Ro5-3335 before, during, and after the development of SuHx-PH in rats to investigate its therapeutic potential. We quantified lung macrophage recruitment and activation in vivo and in vitro in the presence or absence of the RUNX1 inhibitor. We generated conditional VE-cadherin-CreERT2; ZsGreen mice for labelling adult endothelium and lineage tracing in the SuHx-PH model. We also generated conditional Cdh5-CreERT2; Runx1(flox/flox) mice to delete Runx1 gene in adult endothelium and LysM-Cre; Runx1(flox/flox) mice to delete Runx1 gene in cells of myeloid lineage, and then subjected these mice to SuHx-PH induction. RUNX1 inhibition in vivo effectively prevented the development, blocked the progression, and reversed established SuHx-induced PH in rats. RUNX1 inhibition significantly dampened lung macrophage recruitment and activation. Furthermore, lineage tracing with the inducible VE-cadherin-CreERT2; ZsGreen mice demonstrated that a RUNX1-dependent endothelial to haematopoietic transformation occurred during the development of SuHx-PH. Finally, tissue-specific deletion of Runx1 gene either in adult endothelium or in cells of myeloid lineage prevented the mice from developing SuHx-PH, suggesting that RUNX1 is required for the development of PH. CONCLUSION: By blocking RUNX1-dependent endothelial to haematopoietic transformation and pulmonary macrophage recruitment and activation, targeting RUNX1 may be as a novel treatment modality for pulmonary arterial hypertension.

Green tea epigallo-catechin-galleate ameliorates the development of obliterative airway disease.

Lung transplantation has the worst outcome compared to all solid organ transplants due to chronic rejection known as obliterative bronchiolitis (OB). Pathogenesis of OB is a complex interplay of alloimmune-dependent and -independent factors, which leads to the development of inflammation, fibrosis, and airway obliteration that have been resistant to therapy. The alloimmune-independent inflammatory pathway has been the recent focus in the pathogenesis of rejection, suggesting that targeting this may offer therapeutic benefits. As a potent anti-inflammatory agent, epigallo-catechin-galleate (EGCG), a green tea catechin, has been very effective in ameliorating inflammation in a variety of diseases, providing the rationale for its use in this study in a murine heterotopic tracheal allograft model of OB. Mice treated with EGCG had reduced inflammation, with significantly less neutrophil and macrophage infiltration and significantly reduced fibrosis. On further investigation into the mechanisms, inflammatory cytokines keratinocyte (KC), interleukin-17 (IL-17), and tumor necrosis factor-α (TNF-α), involved in neutrophil recruitment, were reduced in the EGCG-treated mice. In addition, monocyte chemokine monocyte chemoattractant protein-1 (MCP-1) was significantly reduced by EGCG treatment. Antifibrotic cytokine interferon-γ-inducible protein-10 (IP-10) was increased and profibrotic cytokine transforming growth factor-ß (TGF-ß) was reduced, further characterizing the antifibrotic effects of EGCG. These findings suggest that EGCG has great potential in ameliorating the development of obliterative airway disease.

Effect of dose, dosing intervals, and hypoxic stress on the reversal of pulmonary hypertension by mesenchymal stem cell extracellular vesicles.

RATIONALE: Mesenchymal stem cell extracellular vesicles (MSC EVs) reverse pulmonary hypertension, but little information is available regarding what dose is effective and how often it needs to be given. This study examined the effects of dose reduction and use of longer dosing intervals and the effect of hypoxic stress of MSC prior to EV collection. METHODS: Adult male rats with pulmonary hypertension induced by Sugen 5416 and three weeks of hypoxia (SuHx-pulmonary hypertension) were injected with MSC EV or phosphate buffered saline the day of removal from hypoxia using one of the following protocols: (1) Once daily for three days at doses of 0.2, 1, 5, 20, and 100 µg/kg, (2) Once weekly (100 µg/kg) for five weeks, (3) Once every other week (100 µg/kg) for 10 weeks, (4) Once daily (20 µg/kg) for three days using EV obtained from MSC exposed to 48 h of hypoxia (HxEV) or MSC kept in normoxic conditions (NxEV). MAIN RESULTS: MSC EV reversed increases in right ventricular systolic pressure (RVSP), right ventricular to left ventricle + septum weight (RV/LV+S), and muscularization index of pulmonary vessels ≤50 µm when given at doses of 20 or 100 µg/kg. RVSP, RV/LV+S, and muscularization index were significantly higher in SuHx-pulmonary hypertension rats treated once weekly with phosphate buffered saline for five weeks or every other week for 10 weeks than in normoxic controls, but not significantly increased in SuHx-pulmonary hypertension rats given MSC EV. Both NxEV and HxEV significantly reduced RVSP, RV/LV+S, and muscularization index, but no differences were seen between treatment groups. CONCLUSIONS: MSC EV are effective at reversing SuHx-pulmonary hypertension when given at lower doses and longer dosing intervals than previously reported. Hypoxic stress does not enhance the efficacy of MSC EV at reversing pulmonary hypertension. These findings support the feasibility of MSC EV as a long-term treatment for pulmonary hypertension.

LRRC15 is an inhibitory receptor blocking SARS-CoV-2 spike-mediated entry in trans.

SARS-CoV-2 infection is mediated by the entry receptor ACE2. Although attachment factors and co-receptors facilitating entry are extensively studied, cellular entry factors inhibiting viral entry are largely unknown. Using a surfaceome CRISPR activation screen, we identified human LRRC15 as an inhibitory receptor for SARS-CoV-2 entry. LRRC15 directly binds to the receptor-binding domain (RBD) of spike protein with a moderate affinity and inhibits spike-mediated entry. Analysis of human lung single cell RNA sequencing dataset reveals that expression of LRRC15 is primarily detected in fibroblasts and particularly enriched in pathological fibroblasts in COVID-19 patients. ACE2 and LRRC15 are not co-expressed in the same cell types in the lung. Strikingly, expression of LRRC15 in ACE2-negative cells blocks spike-mediated viral entry in ACE2+ cell in trans, suggesting a protective role of LRRC15 in a physiological context. Therefore, LRRC15 represents an inhibitory receptor for SARS-CoV-2 regulating viral entry in trans.

Bone marrow stromal cells attenuate lung injury in a murine model of neonatal chronic lung disease.

RATIONALE: Neonatal chronic lung disease, known as bronchopulmonary dysplasia (BPD), remains a serious complication of prematurity despite advances in the treatment of extremely low birth weight infants. OBJECTIVES: Given the reported protective actions of bone marrow stromal cells (BMSCs; mesenchymal stem cells) in models of lung and cardiovascular injury, we tested their therapeutic potential in a murine model of BPD. METHODS: Neonatal mice exposed to hyperoxia (75% O(2)) were injected intravenously on Day 4 with either BMSCs or BMSC-conditioned media (CM) and assessed on Day 14 for lung morphometry, vascular changes associated with pulmonary hypertension, and lung cytokine profile. MEASUREMENTS AND MAIN RESULTS: Injection of BMSCs but not pulmonary artery smooth muscle cells (PASMCs) reduced alveolar loss and lung inflammation, and prevented pulmonary hypertension. Although more donor BMSCs engrafted in hyperoxic lungs compared with normoxic controls, the overall low numbers suggest protective mechanisms other than direct tissue repair. Injection of BMSC-CM had a more pronounced effect than BMSCs, preventing both vessel remodeling and alveolar injury. Treated animals had normal alveolar numbers at Day 14 of hyperoxia and a drastically reduced lung neutrophil and macrophage accumulation compared with PASMC-CM-treated controls. Macrophage stimulating factor 1 and osteopontin, both present at high levels in BMSC-CM, may be involved in this immunomodulation. CONCLUSIONS: BMSCs act in a paracrine manner via the release of immunomodulatory factors to ameliorate the parenchymal and vascular injury of BPD in vivo. Our study suggests that BMSCs and factor(s) they secrete offer new therapeutic approaches for lung diseases currently lacking effective treatment.

Sexual dimorphism in aging hematopoiesis: an earlier decline of hematopoietic stem and progenitor cells in male than female mice.

Adult hematopoietic stem and progenitor cells (HSPCs) reside in the bone marrow (BM) ensuring homeostasis of blood production and immune response throughout life. Sex differences in immunocompetence and mortality are well-documented in humans. However, whether HSPCs behave dimorphically between sexes during aging remains unknown. Here, we show that a significant expansion of BM-derived HSPCs occurs in the middle age of female but in the old age of male mice. We then show that a decline of HSPCs in male mice, as indicated by the expression levels of select hematopoietic genes, occurs much earlier in the aging process than that in female mice. Sex-mismatched heterochronic BM transplantations indicate that the middle-aged female BM microenvironment plays a pivotal role in sustaining hematopoietic gene expression during aging. Furthermore, a higher concentration of the pituitary sex hormone follicle-stimulating hormone (FSH) in the serum and a concomitant higher expression of its receptor on HSPCs in the middle-aged and old female mice than age-matched male mice, suggests that FSH may contribute to the sexual dimorphism in aging hematopoiesis. Our study reveals that HSPCs in the BM niches are possibly regulated in a sex-specific manner and influenced differently by sex hormones during aging hematopoiesis.

BST204, a Rg3 and Rh2 Enriched Ginseng Extract, Upregulates Myotube Formation and Mitochondrial Function in TNF-α-Induced Atrophic Myotubes.

The loss of skeletal muscle mass and function is a serious consequence of chronic diseases and aging. BST204 is a purified ginseng (the root of Panax ginseng) extract that has been processed using ginsenoside-ß-glucosidase and acid hydrolysis to enrich ginsenosides Rg3 and Rh2 from the crude ginseng. BST204 has a broad range of health benefits, but its effects and mechanism on muscle atrophy are currently unknown. In this study, we have examined the effects and underlying mechanisms of BST204 on myotube formation and myotube atrophy induced by tumor necrosis factor-α (TNF-α). BST204 promotes myogenic differentiation and multinucleated myotube formation through Akt activation. BST204 prevents myotube atrophy induced by TNF-α through the activation of Akt/mTOR signaling and down-regulation of muscle-specific ubiquitin ligases, MuRF1, and Atrogin-1. Furthermore, BST204 treatment in atrophic myotubes suppresses mitochondrial reactive oxygen species (ROS) production and regulates mitochondrial transcription factors such as NRF1 and Tfam, through enhancing the activity and expression of peroxisome proliferator-activated receptor-γ coactivator1α (PGC1α). Collectively, our findings indicate that BST204 improves myotube formation and PGC1α-mediated mitochondrial function, suggesting that BST204 is a potential therapeutic or neutraceutical remedy to intervene muscle weakness and atrophy.

Absence of cyclooxygenase-2 exacerbates hypoxia-induced pulmonary hypertension and enhances contractility of vascular smooth muscle cells.

BACKGROUND: Cyclooxygenase-2 (COX-2) is upregulated in pulmonary artery smooth muscle cells (PASMCs) during hypoxia and may play a protective role in the response of the lung to hypoxia. Selective COX-2 inhibition may have detrimental pulmonary vascular consequences during hypoxia. METHODS AND RESULTS: To investigate the role of COX-2 in the pulmonary vascular response to hypoxia, we subjected wild-type and COX-2-deficient mice to a model of chronic normobaric hypoxia. COX-2-null mice developed severe pulmonary hypertension with exaggerated elevation of right ventricular systolic pressure, significant right ventricular hypertrophy, and striking vascular remodeling after hypoxia. Pulmonary vascular remodeling in COX-2-deficient mice was characterized by PASMC hypertrophy but not increased proliferation. Furthermore, COX-2-deficient mice had significant upregulation of the endothelin-1 receptor (ET(A)) in the lung after hypoxia. Similarly, selective pharmacological inhibition of COX-2 in wild-type mice exacerbated hypoxia-induced pulmonary hypertension and resulted in PASMC hypertrophy and increased ET(A) receptor expression in pulmonary arterioles. The absence of COX-2 in vascular smooth muscle cells during hypoxia in vitro augmented traction forces and enhanced contractility of an extracellular matrix. Treatment of COX-2-deficient PASMCs with iloprost, a prostaglandin I(2) analog, and prostaglandin E(2) abrogated the potent contractile response to hypoxia and restored the wild-type phenotype. CONCLUSIONS: Our findings reveal that hypoxia-induced pulmonary hypertension and vascular remodeling are exacerbated in the absence of COX-2 with enhanced ET(A) receptor expression and increased PASMC hypertrophy. COX-2-deficient PASMCs have a maladaptive response to hypoxia manifested by exaggerated contractility, which may be rescued by either COX-2-derived prostaglandin I(2) or prostaglandin E(2).

Loss of lipid phosphatase SHIP1 promotes macrophage differentiation through suppression of dendritic cell differentiation.

SH2-containing inositol 5'-phosphatase-1 (SHIP1) deficiency in mice results in abnormal myeloid expansion, and proinflammatory conditions in the lung. However, the mechanisms involved in SHIP1-mediated regulation of myeloid differentiation remain unclear. Here we show that SHIP1 is a key regulator of early differentiation for dendritic cells (DCs). We also provide critical evidence to modify the function of SHIP1 in in vitro development of BMDCs using the recent framework of defining DCs. We found that loss of SHIP1 suppresses GM-CSF-induced formation of bone marrow-derived DC (BMDC) colonies, leading to reduced BMDC number in BM cell culture. The number of maturated BMDCs decreased in SHIP1-KO culture, due to reduction of immature BMDCs, suggesting SHIP1 is critical for lineage commitment rather than for maturation from myeloid precursors to DCs. We further showed that F4/80+/MHCIIlow BM macrophage-like cells (BMMs) were the main population of SHIP1-KO BM culture. Treatment of wild-type BM culture with 3 α-aminocholestane (3AC), a specific inhibitor for functional activity of SHIP1, caused a similar developmental defect in BMDCs as seen in SHIP1-KO cells, resulting in the absence of BMDC colony, and increased number of BMMs in BM culture. In conclusion, our results suggest that differentiation of BMDCs are markedly impaired under SHIP1 deficient condition, which causes skewed development of myeloid lineage cells manifested as pathological conditions associated with an excess of macrophage population.

Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement.

We previously reported that intramyocardial injection of bone marrow-derived mesenchymal stem cells overexpressing Akt (Akt-MSCs) inhibits ventricular remodeling and restores cardiac function measured 2 wk after myocardial infarction. Here, we report that the functional improvement occurs in < 72 h. This early remarkable effect cannot be readily attributed to myocardial regeneration from the donor cells. Thus, we hypothesized that paracrine actions exerted by the cells through the release of soluble factors might be important mechanisms of tissue repair and functional improvement after injection of the Akt-MSCs. Indeed, in the current study we demonstrate that conditioned medium from hypoxic Akt-MSCs markedly inhibits hypoxia-induced apoptosis and triggers vigorous spontaneous contraction of adult rat cardiomyocytes in vitro. When injected into infarcted hearts, the Akt-MSC conditioned medium significantly limits infarct size and improves ventricular function relative to controls. Support to the paracrine hypothesis is provided by data showing that several genes, coding for factors (VEGF, FGF-2, HGF, IGF-I, and TB4) that are potential mediators of the effects exerted by the Akt-MSC conditioned medium, are significantly up-regulated in the Akt-MSCs, particularly in response to hypoxia. Taken together, our data support Akt-MSC-mediated paracrine mechanisms of myocardial protection and functional improvement.