Hypoxia-induced pulmonary hypertension in adults and newborns: implications for drug development.

Chronic hypoxia-induced pulmonary hypertension (CHPH) presents a complex challenge, characterized by escalating pulmonary vascular resistance and remodeling, threatening both newborns and adults with right heart failure. Despite advances in understanding the pathobiology of CHPH, its molecular intricacies remain elusive, particularly because of the multifaceted nature of arterial remodeling involving the adventitia, media, and intima. Cellular imbalance arises from hypoxia-induced mitochondrial disturbances and oxidative stress, reflecting the diversity in pulmonary hypertension (PH) pathology. In this review, we highlight prominent mechanisms causing CHPH in adults and newborns, and emerging therapeutic targets of potential pharmaceuticals.

Calcium released by osteoclastic resorption stimulates autocrine/paracrine activities in local osteogenic cells to promote coupled bone formation.

A major cause of osteoporosis is impaired coupled bone formation. Mechanistically, both osteoclast-derived and bone-derived growth factors have been previously implicated. Here, we hypothesize that the release of bone calcium during osteoclastic bone resorption is essential for coupled bone formation. Osteoclastic resorption increases interstitial fluid calcium locally from the normal 1.8 mM up to 5 mM. MC3T3-E1 osteoprogenitor cells, cultured in a 3.6 mM calcium medium, demonstrated that calcium signaling stimulated osteogenic cell proliferation, differentiation, and migration. Calcium channel knockdown studies implicated calcium channels, Cav1.2, store-operated calcium entry (SOCE), and calcium-sensing receptor (CaSR) in regulating bone cell anabolic activities. MC3T3-E1 cells cultured in a 3.6 mM calcium medium expressed increased gene expression of Wnt signaling and growth factors platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and bone morphogenic protein-2 (BMP 2). Our coupling model of bone formation, the receptor activator of nuclear factor-κΒ ligand (RANKL)-treated mouse calvaria, confirmed the role of calcium signaling in coupled bone formation by exhibiting increased gene expression for osterix and osteocalcin. Critically, dual immunocytochemistry showed that RANKL treatment increased osterix-positive cells and increased fluorescence intensity of Cav1.2 and CaSR protein expression per osterix-positive cell. The above data established that calcium released by osteoclasts contributed to the regulation of coupled bone formation. CRISPR/Cas-9 knockout of Cav1.2 in osteoprogenitor cells cultured in basal calcium medium caused a >80% decrease in the expression of downstream osteogenic genes, emphasizing the large magnitude of the effect of calcium signaling. Thus, calcium signaling is a major regulator of coupled bone formation.

Loss of APP in mice increases thigmotaxis and is associated with elevated brain expression of IL-13 and IP-10/CXCL10.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that leads to memory loss and is often accompanied by increased anxiety. Although AD is a heterogeneous disease, dysregulation of inflammatory pathways is a consistent event. Interestingly, the amyloid precursor protein (APP), which is the source of the amyloid peptide Aß, is also necessary for the efficient regulation of the innate immune response. Here, we hypothesize that loss of APP function in mice would lead to cognitive loss and anxiety behavior, both of which are typically present in AD, as well as changes in the expression of inflammatory mediators. To test this hypothesis, we performed open field, Y-maze and novel object recognition tests on 12-18-week-old male and female wildtype and AppKO mice to measure thigmotaxis, short-term spatial memory and long-term recognition memory. We then performed a quantitative multiplexed immunoassay to measure levels of 32 cytokines/chemokines associated with AD and anxiety. Our results showed that AppKO mice, compared to wildtype controls, experienced increased thigmotactic behavior but no memory impairments, and this phenotype correlated with increased IP-10 and IL-13 levels. Future studies will determine whether dysregulation of these inflammatory mediators contributes to pathogenesis in AD.

YAP1/TEAD1 upregulate platelet-derived growth factor receptor beta to promote vascular smooth muscle cell proliferation and neointima formation.

We have previously demonstrated that the transcription co-factor yes-associated protein 1 (YAP1) promotes vascular smooth muscle cell (VSMC) de-differentiation. Yet, the role and underlying mechanisms of YAP1 in neointima formation in vivo remain unclear. The goal of this study was to investigate the role of VSMC-expressed YAP1 in vascular injury-induced VSMC proliferation and delineate the mechanisms underlying its action. Experiments employing gain- or loss-of-function of YAP1 demonstrated that YAP1 promotes human VSMC proliferation. Mechanistically, we identified platelet-derived growth factor receptor beta (PDGFRB) as a novel YAP1 target gene that confers the YAP1-dependent hyper-proliferative effects in VSMCs. Furthermore, we identified TEA domain transcription factor 1 (TEAD1) as a key transcription factor that mediates YAP1-dependent PDGFRß expression. ChIP assays demonstrated that TEAD1 is enriched at a PDGFRB gene enhancer. Luciferase reporter assays further demonstrated that YAP1 and TEAD1 co-operatively activate the PDGFRB enhancer. Consistent with these observations, we found that YAP1 expression is upregulated after arterial injury and correlates with PDGFRß expression and VSMC proliferation in vivo. Using a novel inducible SM-specific Yap1 knockout mouse model, we found that the specific deletion of Yap1 in adult VSMCs is sufficient to attenuate arterial injury-induced neointima formation, largely due to inhibited PDGFRß expression and VSMC proliferation. Our study unravels a novel mechanism by which YAP1/TEAD1 promote VSMC proliferation via transcriptional induction of PDGFRß, thereby enhancing PDGF-BB downstream signaling and promoting neointima formation.

Long noncoding RNA NEAT1 (nuclear paraspeckle assembly transcript 1) is critical for phenotypic switching of vascular smooth muscle cells.

In response to vascular injury, vascular smooth muscle cells (VSMCs) may switch from a contractile to a proliferative phenotype thereby contributing to neointima formation. Previous studies showed that the long noncoding RNA (lncRNA) NEAT1 is critical for paraspeckle formation and tumorigenesis by promoting cell proliferation and migration. However, the role of NEAT1 in VSMC phenotypic modulation is unknown. Herein we showed that NEAT1 expression was induced in VSMCs during phenotypic switching in vivo and in vitro. Silencing NEAT1 in VSMCs resulted in enhanced expression of SM-specific genes while attenuating VSMC proliferation and migration. Conversely, overexpression of NEAT1 in VSMCs had opposite effects. These in vitro findings were further supported by in vivo studies in which NEAT1 knockout mice exhibited significantly decreased neointima formation following vascular injury, due to attenuated VSMC proliferation. Mechanistic studies demonstrated that NEAT1 sequesters the key chromatin modifier WDR5 (WD Repeat Domain 5) from SM-specific gene loci, thereby initiating an epigenetic "off" state, resulting in down-regulation of SM-specific gene expression. Taken together, we demonstrated an unexpected role of the lncRNA NEAT1 in regulating phenotypic switching by repressing SM-contractile gene expression through an epigenetic regulatory mechanism. Our data suggest that NEAT1 is a therapeutic target for treating occlusive vascular diseases.

Effect of aging on stem cells.

Pluripotent stem cells have the remarkable self-renewal ability and are capable of differentiating into multiple diverse cells. There is increasing evidence that the aging process can have adverse effects on stem cells. As stem cells age, their renewal ability deteriorates and their ability to differentiate into the various cell types is altered. Accordingly, it is suggested aging-induced deterioration of stem cell functions may play a key role in the pathophysiology of the various aging-associated disorders. Understanding the role of the aging process in deterioration of stem cell function is crucial, not only in understanding the pathophysiology of aging-associated disorders, but also in future development of novel effective stem cell-based therapies to treat aging-associated diseases. This review article first focuses on the basis of the various aging disease-related stem cell dysfunction. It then addresses the several concepts on the potential mechanism that causes aging-related stem cell dysfunction. It also briefly discusses the current potential therapies under development for aging-associated stem cell defects.

Unique Regenerative Mechanism to Replace Bone Lost During Dietary Bone Depletion in Weanling Mice.

The present study was undertaken to determine the mechanism whereby calcitropic hormones and mesenchymal stem cell progeny changes are involved in bone repletion, a regenerative bone process that restores the bone lost to calcium deficiency. To initiate depletion, weanling mice with a mixed C57BL/6 (75%) and CD1 (25%) genetic background were fed a calcium-deficient diet (0.01%) for 14 days. For repletion, the mice were fed a control diet containing 1.2% calcium for 14 days. Depletion decreased plasma calcium and increased plasma parathyroid hormone, 1,25(OH)2D (calcitriol), and C-terminal telopeptide of type I collagen. These plasma parameters quickly returned toward normal on repletion. The trabecular bone volume and connectivity decreased drastically during depletion but were completely restored by the end of repletion. This bone repletion process largely resulted from the development of new bone formation. When bromodeoxyuridine (BrdU) was administered in the middle of depletion for 3 days and examined by fluorescence-activated cell sorting at 7 days into repletion, substantial increases in BrdU incorporation were seen in several CD105 subsets of cells of osteoblastic lineage. When BrdU was administered on days 1 to 3 of repletion and examined 11 days later, no increases in BrdU were seen in these subsets. Additionally, osteocytes that stained positively for BrdU were increased during depletion. In conclusion, the results of the present study have established a unique regenerative mechanism to initiate bone repair during the bone insult. Calcium homeostatic mechanisms and the bone repletion mechanism are opposing functions but are simultaneously orchestrated such that both endpoints are optimized. These results have potential clinical relevance for disease entities such as type 2 osteoporosis.

Cardiac sodium channel regulator MOG1 regulates cardiac morphogenesis and rhythm.

MOG1 was initially identified as a protein that interacts with the small GTPase Ran involved in transport of macromolecules into and out of the nucleus. In addition, we have established that MOG1 interacts with the cardiac sodium channel Nav1.5 and regulates cell surface trafficking of Nav1.5. Here we used zebrafish as a model system to study the in vivo physiological role of MOG1. Knockdown of mog1 expression in zebrafish embryos significantly decreased the heart rate (HR). Consistently, the HR increases in embryos with over-expression of human MOG1. Compared with wild type MOG1 or control EGFP, mutant MOG1 with mutation E83D associated with Brugada syndrome significantly decreases the HR. Interestingly, knockdown of mog1 resulted in abnormal cardiac looping during embryogenesis. Mechanistically, knockdown of mog1 decreases expression of hcn4 involved in the regulation of the HR, and reduces expression of nkx2.5, gata4 and hand2 involved in cardiac morphogenesis. These data for the first time revealed a novel role that MOG1, a nucleocytoplasmic transport protein, plays in cardiac physiology and development.

MicroRNA-15b/16 Attenuates Vascular Neointima Formation by Promoting the Contractile Phenotype of Vascular Smooth Muscle Through Targeting YAP.

OBJECTIVE: To investigate the functional role of the microRNA (miR)-15b/16 in vascular smooth muscle (SM) phenotypic modulation. APPROACH AND RESULTS: We found that miR-15b/16 is one of the most abundant mRs expressed in contractile vascular smooth muscle cells (VSMCs). However, when contractile VSMCs get converted to a synthetic phenotype, miR-15b/16 expression is significantly reduced. Knocking down endogenous miR-15b/16 in VSMCs attenuates SM-specific gene expression but promotes VSMC proliferation and migration. Conversely, overexpression of miR-15b/16 promotes SM contractile gene expression while attenuating VSMC migration and proliferation. Consistent with this, overexpression of miR-15b/16 in a rat carotid balloon injury model markedly attenuates injury-induced SM dedifferentiation and neointima formation. Mechanistically, we identified the potent oncoprotein yes-associated protein (YAP) as a downstream target of miR-15b/16 in VSMCs. Reporter assays validated that miR-15b/16 targets YAP's 3' untranslated region. Moreover, overexpression of miR-15b/16 significantly represses YAP expression, whereas conversely, depletion of endogenous miR-15b/16 results in upregulation of YAP expression. CONCLUSIONS: These results indicate that miR-15b/16 plays a critical role in SM phenotypic modulation at least partly through targeting YAP. Restoring expression of miR-15b/16 would be a potential therapeutic approach for treatment of proliferative vascular diseases.