Evidence That Anemia Accelerates AS Progression Via Shear-Induced TGF-ß1 Activation: Heyde's Syndrome Comes Full Circle.

The severity of aortic stenosis (AS) is associated with acquired von Willebrand syndrome (AVWS) and gastrointestinal bleeding, leading to anemia (Heyde's syndrome). We investigated how anemia is linked with AS and AVWS using the LA100 mouse model and patients with AS. Induction of anemia in LA100 mice increased transforming growth factor (TGF)-ß1 activation, AVWS, and AS progression. Patients age >75 years with severe AS had higher plasma TGF-ß1 levels and more severe anemia than AS patients age <75 years, and there was a correlation between TGF-ß1 and anemia. These data are compatible with the hypothesis that the blood loss anemia of Heyde's syndrome contributes to AS progression via WSS-induced activation of platelet TGF-ß1 and additional gastrointestinal bleeding via WSS-induced AVWS.

Non-Necroptotic Roles of MLKL in Diet-Induced Obesity, Liver Pathology, and Insulin Sensitivity: Insights from a High-Fat, High-Fructose, High-Cholesterol Diet Mouse Model.

Chronic inflammation is a key player in metabolic dysfunction-associated fatty liver disease (MAFLD) progression. Necroptosis, an inflammatory cell death pathway, is elevated in MAFLD patients and mouse models, yet its role is unclear due to the diverse mouse models and inhibition strategies. In our study, we inhibited necroptosis by targeting mixed lineage kinase domain-like pseudokinase (MLKL), the terminal effector of necroptosis, in a high-fat, high-fructose, high-cholesterol (HFHFrHC) mouse model of diet-induced MAFLD. Despite the HFHFrHC diet upregulating MLKL (2.5-fold), WT mice livers showed no increase in necroptosis markers or associated proinflammatory cytokines. Surprisingly, Mlkl-/- mice experienced exacerbated liver inflammation without protection from diet-induced liver damage, steatosis, or fibrosis. In contrast, Mlkl+/- mice showed a significant reduction in these parameters that was associated with elevated Pparα and Pparγ levels. Both Mlkl-/- and Mlkl+/- mice on the HFHFrHC diet resisted diet-induced obesity, attributed to the increased beiging, enhanced oxygen consumption, and energy expenditure due to adipose tissue, and exhibited improved insulin sensitivity. These findings highlight the tissue-specific effects of MLKL on the liver and adipose tissue, and they suggest a dose-dependent effect of MLKL on liver pathology.

Non-Necroptotic Roles of MLKL in Diet-Induced Obesity, Liver Pathology, and Insulin Sensitivity: Insights from a High Fat, High Fructose, High Cholesterol Diet Mouse Model.

Chronic inflammation is a key player in metabolic dysfunction-associated fatty liver disease (MAFLD) progression. Necroptosis, an inflammatory cell death pathway, is elevated in MAFLD patients and mouse models, yet its role is unclear due to diverse mouse models and inhibition strategies. In our study, we inhibited necroptosis by targeting mixed lineage kinase domain like pseudokinase (MLKL), the terminal effector of necroptosis, in a high-fat, high-fructose, high-cholesterol (HFHFrHC) mouse model of diet-induced MAFLD mouse model. Despite HFHFrHC diet upregulating MLKL (2.5-fold), WT mice livers showed no increase in necroptosis markers or associated proinflammatory cytokines. Surprisingly, Mlkl -/- mice experienced exacerbated liver inflammation without protection from diet-induced liver damage, steatosis, or fibrosis. In contrast, Mlkl +/- mice showed significant reduction in these parameters that was associated with elevated Pparα and Pparγ levels. Both Mlkl -/- and Mlkl +/- mice on HFHFrHC diet resisted diet-induced obesity, attributed to increased beiging, enhanced oxygen consumption and energy expenditure due to adipose tissue, and exhibited improved insulin sensitivity. These findings highlight the tissue specific effects of MLKL on the liver and adipose tissue, and suggest a dose-dependent effect of MLKL on liver pathology.

PROX1 Inhibits PDGF-B Expression to Prevent Myxomatous Degeneration of Heart Valves.

BACKGROUND: Cardiac valve disease is observed in 2.5% of the general population and 10% of the elderly people. Effective pharmacological treatments are currently not available, and patients with severe cardiac valve disease require surgery. PROX1 (prospero-related homeobox transcription factor 1) and FOXC2 (Forkhead box C2 transcription factor) are transcription factors that are required for the development of lymphatic and venous valves. We found that PROX1 and FOXC2 are expressed in a subset of valvular endothelial cells (VECs) that are located on the downstream (fibrosa) side of cardiac valves. Whether PROX1 and FOXC2 regulate cardiac valve development and disease is not known. METHODS: We used histology, electron microscopy, and echocardiography to investigate the structure and functioning of heart valves from Prox1ΔVEC mice in which Prox1 was conditionally deleted from VECs. Isolated valve endothelial cells and valve interstitial cells were used to identify the molecular mechanisms in vitro, which were tested in vivo by RNAScope, additional mouse models, and pharmacological approaches. The significance of our findings was tested by evaluation of human samples of mitral valve prolapse and aortic valve insufficiency. RESULTS: Histological analysis revealed that the aortic and mitral valves of Prox1ΔVEC mice become progressively thick and myxomatous. Echocardiography revealed that the aortic valves of Prox1ΔVEC mice are stenotic. FOXC2 was downregulated and PDGF-B (platelet-derived growth factor-B) was upregulated in the VECs of Prox1ΔVEC mice. Conditional knockdown of FOXC2 and conditional overexpression of PDGF-B in VECs recapitulated the phenotype of Prox1ΔVEC mice. PDGF-B was also increased in mice lacking FOXC2 and in human mitral valve prolapse and insufficient aortic valve samples. Pharmacological inhibition of PDGF-B signaling with imatinib partially ameliorated the valve defects of Prox1ΔVEC mice. CONCLUSIONS: PROX1 antagonizes PDGF-B signaling partially via FOXC2 to maintain the extracellular matrix composition and prevent myxomatous degeneration of cardiac valves.

Neonatal intake of Omega-3 fatty acids enhances lipid oxidation in adipocyte precursors.

Establishing metabolic programming begins during fetal and postnatal development, and early-life lipid exposures play a critical role during neonatal adipogenesis. We define how neonatal consumption of a low omega-6 to -3 fatty acid ratio (n6/n3 FA ratio) establishes FA oxidation in adipocyte precursor cells (APCs) before they become adipocytes. In vivo, APCs isolated from mouse pups exposed to the low n6/n3 FA ratio had superior FA oxidation capacity, elevated beige adipocyte mRNAs Ppargc1α, Ucp2, and Runx1, and increased nuclear receptor NR2F2 protein. In vitro, APC treatment with NR2F2 ligand-induced beige adipocyte mRNAs and increased mitochondrial potential but not mass. Single-cell RNA-sequencing analysis revealed low n6/n3 FA ratio yielded more mitochondrial-high APCs and linked APC NR2F2 levels with beige adipocyte signatures and FA oxidation. Establishing beige adipogenesis is of clinical relevance, because fat depots with energetically active, smaller, and more numerous adipocytes improve metabolism and delay metabolic dysfunction.

Infant Red Blood Cell Arachidonic to Docosahexaenoic Acid Ratio Inversely Associates with Fat-Free Mass Independent of Breastfeeding Exclusivity.

The prevalence of childhood obesity has increased nearly ten times over the last 40 years, influenced by early life nutrients that have persistent effects on life-long metabolism. During the first six months, infants undergo accelerated adipose accumulation, but little is known regarding infant fatty acid status and its relationship to infant body composition. We tested the hypothesis that a low arachidonic to docosahexaenoic acid ratio (AA/DHA) in infant red blood cells (RBCs), a long-term indicator of fatty acid intake, would associate with more infant fat-free mass (FFM) and/or less adipose accumulation over the first 4 months of life. The fatty acid and composition of breastmilk and infant RBCs, as well as the phospholipid composition of infant RBCs, were quantified using targeted and unbiased lipid mass spectrometry from infants predominantly breastfed or predominantly formula-fed. Regardless of feeding type, FFM accumulation was inversely associated with the infant's RBC AA/DHA ratio (p = 0.029, R2 = 0.216). Infants in the lowest AA/DHA ratio tertile had significantly greater FFM when controlling for infant sex, adiposity at 2 weeks, and feeding type (p < 0.0001). Infant RBC phospholipid analyses revealed greater peroxisome-derived ether lipids in the low AA/DHA group, primarily within the phosphatidylethanolamines. Our findings support a role for a low AA/DHA ratio in promoting FFM accrual and identify peroxisomal activity as a target of DHA in the growing infant. Both FFM abundance and peroxisomal activity may be important determinants of infant metabolism during development.

Oncogenic Integration of Nucleotide Metabolism via Fatty Acid Synthase in Non-Hodgkin Lymphoma.

Metabolic dysfunctions enabling increased nucleotide biosynthesis are necessary for supporting malignant proliferation. Our investigations indicate that upregulation of fatty acid synthase (FASN) and de novo lipogenesis, commonly observed in many cancers, are associated with nucleotide metabolic dysfunction in lymphoma. The results from our experiments showed that ribonucleotide and deoxyribonucleotide pool depletion, suppression of global RNA/DNA synthesis, and cell cycle inhibition occurred in the presence of FASN inhibition. Subsequently, we observed that FASN inhibition caused metabolic blockade in the rate-limiting step of the oxidative branch of the pentose phosphate pathway (oxPPP) catalyzed by phosphogluconate dehydrogenase (PGDH). Furthermore, we determined that FASN inhibitor treatment resulted in NADPH accumulation and inhibition of PGDH enzyme activity. NADPH is a cofactor utilized by FASN, also a known allosteric inhibitor of PGDH. Through cell-free enzyme assays consisting of FASN and PGDH, we delineated that the PGDH-catalyzed ribulose-5-phosphate synthesis is enhanced in the presence of FASN and is suppressed by increasing concentrations of NADPH. Additionally, we observed that FASN and PGDH were colocalized in the cytosol. The results from these experiments led us to conclude that NADP-NADPH turnover and the reciprocal stimulation of FASN and PGDH catalysis are involved in promoting oxPPP and nucleotide biosynthesis in lymphoma. Finally, a transcriptomic analysis of non-Hodgkin's lymphoma (n = 624) revealed the increased expression of genes associated with metabolic functions interlinked with oxPPP, while the expression of genes participating in oxPPP remained unaltered. Together we conclude that FASN-PGDH enzymatic interactions are involved in enabling oxPPP and nucleotide metabolic dysfunction in lymphoma tumors.

N-Acetylcysteine Inhibits Aortic Stenosis Progression in a Murine Model by Blocking Shear-Induced Activation of Platelet Latent Transforming Growth Factor Beta 1.

Objective: Aortic stenosis (AS) is characterized by narrowing of the aortic valve opening, resulting in peak blood flow velocity that induces high wall shear stress (WSS) across the valve. Severe AS leads to heart failure and death. There is no treatment available for AS other than valve replacement. Platelet-derived transforming growth factor beta 1 (TGF-ß1) partially contributes to AS progression in mice, and WSS is a potent activator of latent TGF-ß1. N-acetylcysteine (NAC) inhibits WSS-induced TGF-ß1 activation in vitro. We hypothesize that NAC will inhibit AS progression by inhibiting WSS-induced TGF-ß1 activation. Approach: We treated a cohort of Ldlr(-/-)Apob(100/100) low density lipoprotein receptor (LDLR) mice fed a high-fat diet with NAC (2% in drinking water) at different stages of disease progression and measured its effect on AS progression and TGF-ß1 activation. Results: Short-term NAC treatment inhibited AS progression in mice with moderate and severe AS relative to controls, but not in LDLR mice lacking platelet-derived TGF-ß1 (TGF-ß1platlet-KO-LDLR). NAC treatment reduced TGF-ß signaling, p-Smad2 and collagen levels, and mesenchymal transition from isolectin B4 and CD45-positive cells in LDLR mice. Mechanistically, NAC treatment resulted in plasma NAC concentrations ranging from 75.5 to 449.2 ng/mL, which were sufficient to block free thiol labeling of plasma proteins and reduce active TGF-ß1 levels without substantially affecting reactive oxygen species-modified products in valvular cells. Conclusions: Short-term treatment with NAC inhibits AS progression by inhibiting WSS-induced TGF-ß1 activation in the LDLR mouse model of AS, motivating a clinical trial of NAC and/or other thiol-reactive agent(s) as a potential therapy for AS.

Myocardial Hypertrophy and Compensatory Increase in Systolic Function in a Mouse Model of Oxidative Stress.

Free radicals, or reactive oxygen species, have been implicated as one of the primary causes of myocardial pathologies elicited by chronic diseases and age. The imbalance between pro-oxidants and antioxidants, termed "oxidative stress", involves several pathological changes in mouse hearts, including hypertrophy and cardiac dysfunction. However, the molecular mechanisms and adaptations of the hearts in mice lacking cytoplasmic superoxide dismutase (Sod1KO) have not been investigated. We used echocardiography to characterize cardiac function and morphology in vivo. Protein expression and enzyme activity of Sod1KO were confirmed by targeted mass spectrometry and activity gel. The heart weights of the Sod1KO mice were significantly increased compared with their wildtype peers. The increase in heart weights was accompanied by concentric hypertrophy, posterior wall thickness of the left ventricles (LV), and reduced LV volume. Activated downstream pathways in Sod1KO hearts included serine-threonine kinase and ribosomal protein synthesis. Notably, the reduction in LV volume was compensated by enhanced systolic function, measured by increased ejection fraction and fractional shortening. A regulatory sarcomeric protein, troponin I, was hyper-phosphorylated in Sod1KO, while the vinculin protein was upregulated. In summary, mice lacking cytoplasmic superoxide dismutase were associated with an increase in heart weights and concentric hypertrophy, exhibiting a pathological adaptation of the hearts to oxidative stress.

HIV protease inhibitor ritonavir induces renal fibrosis and dysfunction: role of platelet-derived TGF-ß1 and intervention via antioxidant pathways.

OBJECTIVE: Chronic kidney disease (CKD) with tubular injury and fibrosis occurs in HIV infection treated with certain protease inhibitor-based antiretroviral therapies. The pathophysiology is unclear. DESIGN: We hypothesized that fibrosis, mediated by platelet-derived transforming growth factor (TGF)-ß1, underlies protease inhibitor-associated CKD. We induced this in mice exposed to the protease inhibitor ritonavir (RTV), and intervened with low-dose inhaled carbon monoxide (CO), activating erythroid 2-related factor (Nrf2)-associated antioxidant pathways. METHODS: Wild-type C57BL/6 mice and mice deficient in platelet TGF-ß1, were given RTV (10 mg/kg) or vehicle daily for 8 weeks. Select groups were exposed to CO (250 ppm) for 4 h after RTV or vehicle injection. Renal disorder, fibrosis, and TGF-ß1-based and Nrf2-based signaling were examined by histology, immunofluorescence, and flow cytometry. Renal damage and dysfunction were assessed by KIM-1 and cystatin C ELISAs. Clinical correlations were sought among HIV-infected individuals. RESULTS: RTV-induced glomerular and tubular injury, elevating urinary KIM-1 (P = 0.004). It enhanced TGF-ß1-related signaling, accompanied by kidney fibrosis, macrophage polarization to an inflammatory phenotype, and renal dysfunction with cystatin C elevation (P = 0.008). Mice lacking TGF-ß1 in platelets were partially protected from these abnormalities. CO inhibited RTV-induced fibrosis and macrophage polarization in association with upregulation of Nrf2 and heme oxygenase-1 (HO-1). Clinically, HIV infection correlated with elevated cystatin C levels in untreated women (n = 17) vs. age-matched controls (n = 19; P = 0.014). RTV-treated HIV+ women had further increases in cystatin C (n = 20; P = 0.05), with parallel elevation of HO-1. CONCLUSION: Platelet TGF-ß1 contributes to RTV-induced kidney fibrosis and dysfunction, which may be amenable to antioxidant interventions.

Oscillatory shear potentiates latent TGF-ß1 activation more than steady shear as demonstrated by a novel force generator.

Cardiovascular mechanical stresses trigger physiological and pathological cellular reactions including secretion of Transforming Growth Factor ß1 ubiquitously in a latent form (LTGF-ß1). While complex shear stresses can activate LTGF-ß1, the mechanisms underlying LTGF-ß1 activation remain unclear. We hypothesized that different types of shear stress differentially activate LTGF-ß1. We designed a custom-built cone-and-plate device to generate steady shear (SS) forces, which are physiologic, or oscillatory shear (OSS) forces characteristic of pathologic states, by abruptly changing rotation directions. We then measured LTGF-ß1 activation in platelet releasates. We modeled and measured flow profile changes between SS and OSS by computational fluid dynamics (CFD) simulations. We found a spike in shear rate during abrupt changes in rotation direction. OSS activated TGF-ß1 levels significantly more than SS at all shear rates. OSS altered oxidation of free thiols to form more high molecular weight protein complex(es) than SS, a potential mechanism of shear-dependent LTGF-ß1 activation. Increasing viscosity in platelet releasates produced higher shear stress and higher LTGF-ß1 activation. OSS-generated active TGF-ß1 stimulated higher pSmad2 signaling and endothelial to mesenchymal transition (EndoMT)-related genes PAI-1, collagen, and periostin expression in endothelial cells. Overall, our data suggest variable TGF-ß1 activation and signaling occurs with competing blood flow patterns in the vasculature to generate complex shear stress, which activates higher levels of TGF-ß1 to drive vascular remodeling.

Inactivation of platelet-derived TGF-ß1 attenuates aortic stenosis progression in a robust murine model.

Aortic stenosis (AS) is a degenerative heart condition characterized by fibrosis and narrowing of aortic valves (AV), resulting in high wall shear stress (WSS) across valves. AS is associated with high plasma levels of transforming growth factor-ß1 (TGF-ß1), which can be activated by WSS to induce organ fibrosis, but the cellular source of TGF-ß1 is not clear. Here, we show that platelet-derived TGF-ß1 plays an important role in AS progression. We first established an aggressive and robust murine model of AS, using the existing Ldlr -/- Apob100/100 (LDLR) breed of mice, and accelerated AS progression by feeding them a high-fat diet (HFD). We then captured very high resolution images of AV movement and thickness and of blood flow velocity across the AV, using a modified ultrasound imaging technique, which revealed early evidence of AS and distinguished different stages of AS progression. More than 90% of LDLR animals developed AS within 6 months of HFD. Scanning electron microscopy and whole-mount immunostaining imaging of AV identified activated platelets physically attached to valvular endothelial cells (VEC) expressing high phosphorylated Smad2 (p-Smad2). To test the contribution of platelet-derived TGF-ß1 in AS, we derived LDLR mice lacking platelet TGF-ß1 (TGF-ß1platelet-KO-LDLR) and showed reduced AS progression and lower p-Smad2 and myofibroblasts in their AV compared with littermate controls fed the HFD for 6 months. Our data suggest that platelet-derived TGF-ß1 triggers AS progression by inducing signaling in VEC, and their subsequent transformation into collagen-producing-myofibroblasts. Thus, inhibiting platelet-derived TGF-ß1 might attenuate or prevent fibrotic diseases characterized by platelet activation and high WSS, such as AS.

Platelet TGF-ß1 deficiency decreases liver fibrosis in a mouse model of liver injury.

Transforming growth factor-ß1 (TGF-ß1) signaling in hepatic stellate cells (HSCs) plays a primary role in liver fibrosis, but the source of TGF-ß1 is unclear. Because platelets are rich in TGF-ß1, we examined the role of platelet TGF-ß1 in liver fibrosis by challenging wild-type (WT) mice and mice deficient in platelet TGF-ß1 (PF4CreTgfb1f/f) with carbon tetrachloride (CCl4), an inducer of acute hepatic injury and chronic fibrosis. CCl4 elicited equivalent hepatic injury in WT and PF4CreTgfb1f/f mice based on loss of cytochrome P450 (Cyp2e1) expression, observed at 6 hours and peaking at 3 days after CCl4 challenge; PF4CreTgfb1f/f mice exhibited less liver fibrosis than control mice. Activated platelets were observed during acute liver injury (6 hours), and WT mice with transient platelet depletion (thrombocytopenia) were partially protected from developing fibrosis compared with control mice (P = .01), suggesting an association between platelet activation and fibrosis. Transient increases in TGF-ß1 levels and Smad2 phosphorylation signaling were observed 6 hours and 3 days, respectively, after CCl4 challenge in WT, but not PF4CreTgfb1f/f , mice, suggesting that increased TGF-ß1 levels originated from platelet-released TGF-ß1 during the initial injury. Numbers of collagen-producing HSCs and myofibroblasts were higher at 3 days and 36 days, respectively, in WT vs PF4CreTgfb1f/f mice, suggesting that platelet TGF-ß1 may have stimulated HSC transdifferentiation into myofibroblasts. Thus, platelet TGF-ß1 partially contributes to liver fibrosis, most likely by initiating profibrotic signaling in HSCs and collagen synthesis. Further studies are required to evaluate whether blocking platelet and TGF-ß1 activation during acute liver injury prevents liver fibrosis.

New methodologies to accurately assess circulating active transforming growth factor-ß1 levels: implications for evaluating heart failure and the impact of left ventricular assist devices.

Transforming growth factor-ß1 (TGF-ß1) has been used as a biomarker in disorders associated with pathologic fibrosis. However, plasma TGF-ß1 assessment is confounded by the significant variation in reported normal values, likely reflecting variable release of the large pool of platelet TGF-ß1 after blood drawing. Moreover, current assays measure only total TGF-ß1, which is dominated by the latent form of TGF-ß1 rather than the biologically active form. To address these challenges, we developed methodologies to prevent ex vivo release of TGF-ß1 and to quantify active TGF-ß1. We then used these techniques to measure TGF-ß1 in healthy controls and patients with heart failure (HF) before and after insertion of left ventricular assist devices (LVAD). Total plasma TGF-ß1 was 1.0 ± 0.60 ng/mL in controls and 3.76 ± 1.55 ng/mL in subjects with HF (P < 0.001), rising to 5.2 ± 2.3 ng/mL following LVAD placement (P = 0.006). These results were paralleled by the active TGF-ß1 values; controls had 3-16 pg/mL active TGF-ß1, whereas levels were 2.7-fold higher in patients with HF before, and 4.2-fold higher after, LVAD implantation. Total TGF-ß1 correlated with levels of the platelet-derived protein thrombospondin-1 (r = 0.87; P < 0.001), suggesting that plasma TGF-ß1 may serve as a surrogate indicator of in vivo platelet activation. von Willebrand factor high molecular weight multimers correlated inversely with TGF-ß1 levels (r = -0.63; P = 0.023), suggesting a role for shear forces in loss of these multimers and platelet activation. In conclusion, accurate assessment of circulating TGF-ß1 may provide a valuable biomarker for in vivo platelet activation and thrombotic disorders.

HIV protease inhibitor-induced cardiac dysfunction and fibrosis is mediated by platelet-derived TGF-ß1 and can be suppressed by exogenous carbon monoxide.

Human immunodeficiency virus (HIV) infection is an independent risk factor for cardiovascular disease. This risk is magnified by certain antiretrovirals, particularly the protease inhibitor ritonavir, but the pathophysiology of this connection is unknown. We postulated that a major mechanism for antiretroviral-associated cardiac disease is pathologic fibrosis linked to platelet activation with release and activation of transforming growth factor (TGF)-ß1, and that these changes could be modeled in a murine system. We also sought to intervene utilizing inhaled carbon monoxide (CO) as proof-of-concept for therapeutics capable of regulating TGF-ß1 signaling and collagen autophagy. We demonstrate decreased cardiac function indices, including cardiac output, ejection fraction and stroke volume, and prominent cardiac fibrosis, in mice exposed to pharmacological doses of ritonavir. Cardiac output and fibrosis correlated with plasma TGF-ß1 levels. Mice with targeted deletion of TGF-ß1 in megakaryocytes/platelets (PF4CreTgfb1flox/flox) were partially protected from ritonavir-induced cardiac dysfunction and fibrosis. Inhalation of low dose CO (250ppm), used as a surrogate for upregulation of inducible heme oxygenase/endogenous CO pathways, suppressed ritonavir-induced cardiac fibrosis. This occurred in association with modulation of canonical (Smad2) and non-canonical (p38) TGF-ß1 signaling pathways. In addition, CO treatment suppressed the M1 pro-inflammatory subset of macrophages and increased M2c regulatory cells in the hearts of RTV-exposed animals. The effects of CO were dependent upon autophagy as CO did not mitigate ritonavir-induced fibrosis in autophagy-deficient LC3-/- mice. These results suggest that platelet-derived TGF-ß1 contributes to ritonavir-associated cardiac dysfunction and fibrosis, extending the relevance of our findings to other antiretrovirals that also activate platelets. The anti-fibrotic effects of CO are linked to alterations in TGF-ß1 signaling and autophagy, suggesting a proof-of-concept for novel interventions in HIV/antiretroviral therapy-mediated cardiovascular disease.

Monocrotaline-Induced Pulmonary Hypertension Involves Downregulation of Antiaging Protein Klotho and eNOS Activity.

The objective of this study is to investigate whether stem cell delivery of secreted Klotho (SKL), an aging-suppressor protein, attenuates monocrotaline-induced pulmonary vascular dysfunction and remodeling. Overexpression of SKL in mesenchymal stem cells (MSCs) was achieved by transfecting MSCs with lentiviral vectors expressing SKL-green fluorescent protein (GFP). Four groups of rats were treated with monocrotaline, whereas an additional group was given saline (control). Three days later, 4 monocrotaline-treated groups received intravenous delivery of nontransfected MSCs, MSC-GFP, MSC-SKL-GFP, and PBS, respectively. Ex vivo vascular relaxing responses to acetylcholine were diminished in small pulmonary arteries (PAs) in monocrotaline-treated rats, indicating pulmonary vascular endothelial dysfunction. Interestingly, delivery of MSCs overexpressing SKL (MSC-SKL-GFP) abolished monocrotaline-induced pulmonary vascular endothelial dysfunction and PA remodeling. Monocrotaline significantly increased right ventricular systolic blood pressure, which was attenuated significantly by MSC-SKL-GFP, indicating improved PA hypertension. MSC-SKL-GFP also attenuated right ventricular hypertrophy. Nontransfected MSCs slightly, but not significantly, improved PA hypertension and pulmonary vascular endothelial dysfunction. MSC-SKL-GFP attenuated monocrotaline-induced inflammation, as evidenced by decreased macrophage infiltration around PAs. MSC-SKL-GFP increased SKL levels, which rescued the downregulation of SIRT1 (Sirtuin 1) expression and endothelial NO synthase (eNOS) phosphorylation in the lungs of monocrotaline-treated rats. In cultured endothelial cells, SKL abolished monocrotaline-induced downregulation of eNOS activity and NO levels and enhanced cell viability. Therefore, stem cell delivery of SKL is an effective therapeutic strategy for pulmonary vascular endothelial dysfunction and PA remodeling. SKL attenuates monocrotaline-induced PA remodeling and PA smooth muscle cell proliferation, likely by reducing inflammation and restoring SIRT1 levels and eNOS activity.

Placenta growth factor and vascular endothelial growth factor a have differential, cell-type specific patterns of expression in vascular cells.

OBJECTIVE: PLGF, a VEGF-A related protein, mediates collateral enlargement via monocytes but plays little role in capillary proliferation. In contrast, VEGF-A mediates both collateral enlargement and capillary proliferation. PLGF has been less thoroughly studied than VEGF-A, and questions remain regarding its regulation and function. Therefore, our goal was to characterize the expression of PLGF by vascular cells. We hypothesized that vascular SMC would express more PLGF than EC, since VEGF-A is primarily expressed by non-EC. METHODS: We compared PLGF and VEGF-A across eight EC and SMC lines, then knocked down PLGF and evaluated cell function. We also assessed the effect of hypoxia on PLGF expression and promoter activity. RESULTS: PLGF was most highly expressed in EC, whereas VEGF-A was most highly expressed in SMC. PLGF knockdown did not affect EC number, migration, or tube formation, but reduced monocyte migration toward EC. Monocyte migration was rescued by exogenous PLGF. Hypoxia increased PLGF protein without activating PLGF gene transcription. CONCLUSIONS: PLGF and VEGF-A have distinct patterns of expression in vascular cells. EC derived PLGF may function primarily in communication between EC and circulating cells. Hypoxia increases EC PLGF expression posttranscriptionally.

Transplantable delivery systems for in situ controlled release of bisphosphonate in orthopedic therapy.

IMPORTANCE OF THE FIELD: Bisphosphonates (BPs), structurally similar to pyrophosphates and functionally superior in restraining osteoclast-induced bone resorption, have been widely used as clinical drugs in the treatment of osteoporosis, bone voids and associated inflammation. However, owing to their high aqueous solubility and the consequently high rate of loss during oral administration, the loading and targeting of BPs pose major challenges in practice. Alternative delivery routes such as nasal, subcutaneous/intramuscular injection have contributed little to improving the bioavailiability and efficacy of BPs. To improve and optimize the delivery efficiency and efficacy of BPs, numerous strategies have been developed and adopted. Studies on controlled release of BPs provide important information on the fabrication of BP delivery systems for in situ treatment. As BPs play an important therapeutic role in osteoporosis and similar diseases, it has become essential and vital to survey various reported fabrication methodologies of these systems and the consequential orthopedic treatments so as to keep abreast with advances in their clinical use. AREAS COVERED IN THIS REVIEW: Transplantable delivery systems for controlled release of BP are reviewed from literature published since 2000. The fabrication pathways and the release of BPs from various material systems are discussed in case studies. Recent progress in CaP models based on the strong and specific chelation between BPs and calcium phosphate crystals is highlighted. WHAT THE READER WILL GAIN: This review offers an outline of the advances in BP controlled release and delivery systems for orthopedic therapy. TAKE HOME MESSAGE: Understanding the cutting-edge BP controlled release and delivery systems for in situ treatment is key to the successful design of a more promising and perfect delivery system for orthopedic therapy. Moreover, developing such delivery systems incorporating the numerous advantages of BPs and controlled release environment requires substantially more flexible models to control better the fate of BP drugs.

Chondrogenesis of synovium-derived mesenchymal stem cells in gene-transferred co-culture system.

A co-culture strategy has been developed in this study wherein rabbit synovial mesenchymal stem cells (SMSCs) are co-cultured with growth factor (GF) transfected articular chondrocytes. Toward this end, both SMSCs and early passage rabbit articular chondrocytes that had been adenovirally transduced with transforming growth factor-beta 3 (TGF-beta3) gene were separately encapsulated in alginate beads and co-cultured in the same pool of chondrogenic medium. The chondrocytes act as transfected companion cells (TCCs) providing GF supply to induce chondrogenic differentiation of SMSCs that play the role of therapeutic progenitor cells (TPCs). Against the same TCC based TGF-beta3 release profile, the co-culture was started at different time points (Day 0, Day 10 and Day 20) but made to last for identical periods of exposure (30 days) so that the exposure conditions could be optimized in terms of initiation and duration. Transfection of TCCs prevents the stem cell based TPCs from undergoing the invasive procedure. It also prevents unpredictable complications in the TPCs caused by long-term constitutive over-expression of a GF. The adenovirally transfected TCCs exhibit a transient GF expression which results in a timely termination of GF supply to the TPCs. The TCC-sourced transgenic TGF-beta3 successfully induced chondrogenesis in the TPCs. Real-time PCR results show enhanced expression of cartilage markers and immuno/histochemical staining for Glycosaminoglycans (GAG) and Collagen II also shows abundant extracellular matrix (ECM) production and chondrogenic morphogenesis in the co-cultured TPCs. These results confirm the efficacy of directing stem cell differentiation towards chondrogenesis and cartilage tissue formation by co-culturing them with GF transfected chondrocytes.