Pelargonic acid vanillylamide alleviates hepatic autophagy and ER stress in hepatic steatosis model.

Pelargonic acid vanillylamide (PAVA) has been shown to reduce hepatic lipid accumulation in an obese rat model, however the underlying mechanism responsible for regulating lipid metabolism remains unclear. This study investigated the molecular mechanisms invoked by PAVA in regulating lipogenesis, autophagy, and endoplasmic reticulum (ER) stress in obese rats. Male Sprague-Dawley rats were fed on a diet consisting of 65.26% fat (16 weeks) and HepG2 cells were incubated with 200 µM oleic acid (OA) plus 100 µM palmitic acid (PA) for 48 h. These treatments resulted in a steatosis model. PAVA was shown to reduce fat deposition in hepatocytes in HepG2 by reducing lipotoxicity, the triglyceride content, the expression of sterol regulatory element binding protein 1c (SREBP-1c) and fatty acid synthase (FASN). PAVA also significantly reduced the calcium level and the expression of calpain 2 and upregulated the expression of Atg7 in comparison to the HFD group. In addition, PAVA was shown to significantly decrease the expression of autophagy pathway-related proteins including LC3 and p62. Treatment with PAVA (1 mg/day) reduced the expressions of ER stress markers Bip, ATF6 (p50), p-IRE1/IRE1, p-eIF2α/eIF2α, pJNK, CHOP and cleaved CASP12. In conclusion, PAVA ameliorated obesity induced hepatic steatosis by attenuating defective autophagy and ER stress pathways.

The capsaicinoid nonivamide suppresses the inflammatory response and attenuates the progression of steatosis in a NAFLD-rat model.

Nonalcoholic fatty liver disease (NAFLD) is relatively associated with comorbidities in obesity and metabolic inflammation. Low-grade inflammation following the high-fat diet (HFD)-induced NAFLD can promote the development of nonalcoholic steatohepatitis (NASH) through particularly liver-resident immune cell recruitment and hepatic nuclear factor kappa B (NF-κB) pathway. Therefore, inflammatory intervention may contribute to NASH reduction. Pelargonic acid vanillylamide (PAVA) or nonivamide is one of the pungent capsaicinoids of Capsicum species and has been found in chili peppers. Our previous study demonstrated that PAVA improved hepatic function, decreased oxidative stress and reduced apoptotic cell death but the insight role of PAVA on NAFLD is still unclear. Thus, this study aimed to investigate the underlying anti-inflammatory mechanism of PAVA in an NAFLD-rat model. Male Sprague Dawley rats were fed with normal diet or HFD for 16 weeks. Then high-fat rats were given vehicle or PAVA (1 mg/kg/day) for another 4 weeks. We found that PAVA alleviated hepatic inflammation associated with the reducing toll-like receptor 4/NF-κB pathway, showing significantly lower recruitment of cluster of differentiation 44. PAVA also maintained activity of insulin signaling pathway, and attenuated NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome formation. NAFLD progresses to NASH through transforming growth factor (TGF-ß1), and also recovery to simple stage followed by PAVA suppresses pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-1ß, interleukin-6, and Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway. Therefore, our findings suggest that PAVA provides a novel therapeutic approach for NAFLD and slows the progression to NASH.

Protective Effect of Neferine in Permanent Cerebral Ischemic Rats via Anti-Oxidative and Anti-Apoptotic Mechanisms.

Permanent cerebral ischemia is a consequence of prolonged cerebral artery occlusion that results in severe brain damage. Neurotoxicity occurring after ischemia can induce brain tissue damage by destroying cell organelles and their function. Neferine is a natural compound isolated from the seed embryos of the lotus plant and has broad pharmacological effects, including blockading of the calcium channels, anti-oxidative stress, and anti-apoptosis. This study investigated the ability of neferine to reduce brain injury after permanent cerebral occlusion. Permanent cerebral ischemia in rats was induced by instigation of occlusion of the middle cerebral artery for 24 h. The rats were divided into 6 groups: sham, permanent middle cerebral artery occlusion (pMCAO), pMCAO with neferine and nimodipine treatment. To investigate the severity of the injury, the neurological deficit score and morphological alterations were investigated. After 24 h, the rats were evaluated to assess neurological deficit, infarct volume, morphological change, and the number of apoptotic cell deaths. In addition, the brain tissues were examined by western blot analysis to calculate the expression of proteins related to oxidative stress and apoptosis. The data showed that the neurological deficit scores and the infarct volume were significantly reduced in the neferine-treated rats compared to the vehicle group. Treatment with neferine significantly reduced oxidative stress with a measurable decrease in 4-hydroxynonenal (4-HNE), nitric oxide (NO), neuronal nitric oxide (nNOS), and calcium levels and an upregulation of Hsp70 expression. Neferine treatment also significantly decreased apoptosis, with a decrease in Bax and cleaved caspase-3 and an increase in Bcl-2. This study suggested that neferine had a neuroprotective effect on permanent cerebral ischemia in rats by diminishing oxidative stress and apoptosis.

Cardiac fibroblasts regulate the development of heart failure via Htra3-TGF-ß-IGFBP7 axis.

Tissue fibrosis and organ dysfunction are hallmarks of age-related diseases including heart failure, but it remains elusive whether there is a common pathway to induce both events. Through single-cell RNA-seq, spatial transcriptomics, and genetic perturbation, we elucidate that high-temperature requirement A serine peptidase 3 (Htra3) is a critical regulator of cardiac fibrosis and heart failure by maintaining the identity of quiescent cardiac fibroblasts through degrading transforming growth factor-ß (TGF-ß). Pressure overload downregulates expression of Htra3 in cardiac fibroblasts and activated TGF-ß signaling, which induces not only cardiac fibrosis but also heart failure through DNA damage accumulation and secretory phenotype induction in failing cardiomyocytes. Overexpression of Htra3 in the heart inhibits TGF-ß signaling and ameliorates cardiac dysfunction after pressure overload. Htra3-regulated induction of spatio-temporal cardiac fibrosis and cardiomyocyte secretory phenotype are observed specifically in infarct regions after myocardial infarction. Integrative analyses of single-cardiomyocyte transcriptome and plasma proteome in human reveal that IGFBP7, which is a cytokine downstream of TGF-ß and secreted from failing cardiomyocytes, is the most predictable marker of advanced heart failure. These findings highlight the roles of cardiac fibroblasts in regulating cardiomyocyte homeostasis and cardiac fibrosis through the Htra3-TGF-ß-IGFBP7 pathway, which would be a therapeutic target for heart failure.

Interplay between HTRA1 and classical signalling pathways in organogenesis and diseases.

The high temperature requirement factor A1 (HTRA1) is a serine protease which modulates an array of signalling pathways driving basal biological processes. HTRA1 plays a significant role in cell proliferation, migration and fate determination, in addition to controlling protein aggregates through refolding, translocation or degradation. The mutation of HTRA1 has been implicated in a plethora of disorders and this has also led to its growing interest as drug therapy target. This review details the involvement of HTRA1 in certain signalling pathways, namely the transforming growth factor beta (TGF-ß), canonical Wingless/Integrated (WNT) and NOTCH signalling pathways during organogenesis and various disease pathogenesis such as preeclampsia, age-related macular degeneration (AMD), small vessel disease and cancer. We have also explored possible avenues of exploiting the serine proteases for therapeutic management of these disorders.

Heterozygous loss of Zbtb38 leads to early embryonic lethality via the suppression of Nanog and Sox2 expression.

OBJECTIVES: Mammalian DNA methyltransferases are essential to re-establish global DNA methylation patterns during implantation, which is critical for transmitting epigenetic information to the next generation. In contrast, the significance of methyl-CpG binding proteins (MBPs) that bind methylated CpG remains almost unknown at this stage. We previously demonstrated that Zbtb38 (also known as CIBZ)-a zinc finger type of MBP-is required for mouse embryonic stem (ES) cell proliferation by positively regulating Nanog expression. However, the physiological function of Zbtb38 in vivo remains unclear. MATERIALS AND METHODS: This study used the Cre-loxP system to generate conditional Zbtb38 knockout mice. Cell proliferation and apoptosis were studied by immunofluorescence staining. Quantitative real-time PCR, immunoblotting and immunofluorescence were performed to investigate the molecular mechanisms. RESULTS: Germline loss of the Zbtb38 single allele resulted in decreased epiblast cell proliferation and increased apoptosis shortly after implantation, leading to early embryonic lethality. Heterozygous loss of Zbtb38 reduced the expression of Nanog, Sox2, and the genes responsible for epiblast proliferation, differentiation, and cell viability. Although this early lethal phenotype, Zbtb38 is dispensable for ES cell establishment and identity. CONCLUSIONS: These findings indicate that Zbtb38 is essential for early embryonic development via the suppression of Nanog and Sox2 expression.

rs10737680 polymorphism in complement factor H and neovascular age-related macular degeneration in Yogyakarta, Indonesia.

Background: Neovascular age-related macular degeneration (nAMD) is one of the main causes of blindness in developed countries. Complement factor H (CFH) is one of the genes involved in the pathogenesis of nAMD. This study investigated the rs10737680 polymorphism in CFH and its conferred susceptibility to nAMD in Yogyakarta, Indonesia. Methods: This case-control hospital-based study recruited participants consisting of 96 patients with nAMD and 101 controls without nAMD from the Eye Polyclinic of Sardjito Hospital, YAP Eye Hospital, and Hardjolukito Hospital Yogyakarta. nAMD was diagnosed when fundus examination, fundus photographs, and optical coherence tomography revealed hard or soft drusen in the macular area measuring > 63 µm that appeared below the retinal pigment epithelium, with or without macular hypo- or hyperpigmentation, and was accompanied by choroidal neovascularization. Genomic DNA was extracted using a commercial DNA isolation kit. The restriction fragment length polymorphism technique was used to identify the rs10737680 polymorphism in CFH. Results: The mean (standard deviation [SD]) age of the nAMD group was not homogeneous with that of the control group (P < 0.05); 65.41 (9.74) years versus 68.24 (7.82) years. The number of patients with hypertension in the nAMD group was significantly higher than in the control group (P < 0.05). In the nAMD group, the genotype distribution indicated homozygous risk allele in 34.38%, heterozygous risk allele in 57.29%, and homozygous non-risk allele in 8.33%. In the control group, the genotype distribution indicated homozygous risk allele in 21.78%, heterozygous risk allele in 36.63%, and homozygous non-risk allele in 41.58%. Statistical analysis between the two study groups according to homozygous risk allele genotype (odds ratio [OR], 7.87; 95% confidence interval [CI], 2.88-22.79) and heterozygous genotype (OR, 7.80; 95% CI, 3.11-21.19) showed a significant difference (both P < 0.01). Conclusions: Homozygous risk allele was less frequent than heterogeneous risk allele in patients with nAMD; however, both increased the risk for nAMD. Although the homozygous or heterozygous risk-alleles were detected in most patients, yet other important genetic or environmental factors could be involved in the pathogenesis of nAMD. Overall, we found a significant association between rs10737680 polymorphism in CFH and the susceptibility to nAMD in Yogyakarta, Indonesia; however, future studies are needed to fully delineate the mechanism.

Association of the HtrA1 rs11200638 Polymorphism with Neovascular Age-Related Macular Degeneration in Indonesia.

INTRODUCTION: The aim of this study was to investigate the association of the HtrA1 rs11200638 polymorphism with neovascular age-related macular degeneration (nAMD) in Indonesia. METHODS: This case-control study included 80 patients with nAMD and 85 controls. Demographic parameters and whole blood were collected from each participant. Genomic DNA was extracted and used to assess the rs11200638 genotype by PCR and restriction enzyme digestion. Associations between the HtrA1 rs11200638 polymorphism and other risk factors for susceptibility to nAMD were assessed using the logistic regression model. RESULTS: Significant allelic associations between the HtrA1 polymorphism and nAMD were detected (odds ratio [OR] 8.67; 95% confidence interval [CI] 4.88-15.41; P < 0.001). Genotype analysis showed a statistical difference between the nAMD group and the control group (P < 0.001). In the multiple adjusted logistic regression model, people with the AA genotype were more likely to have nAMD although there was a wide confidence interval (OR 19.65; 95% CI 4.52-85.38; P < 0.001). CONCLUSION: Our findings show that the risk of nAMD increased in the presence of risk alleles of HtrA1 rs11200638.

Neferine Protects Against Brain Damage in Permanent Cerebral Ischemic Rat Associated with Autophagy Suppression and AMPK/mTOR Regulation.

Neferine is the major alkaloid compound isolated from the seed embryos of lotus. Neferine has many pharmacological effects, such as anti-inflammatory, antioxidative stress, and antiapoptotic effects, and it maintains autophagic balance. The purpose of this study was to explore the mechanism by which neferine attenuates autophagy after permanent cerebral ischemia in rats. We performed permanent cerebral ischemia in rats by middle cerebral artery occlusion (pMCAO) for 12 h with or without administration of neferine or nimodipine, a calcium (Ca2+) channel blocker. Neuroprotective effects were determined by evaluating the infarct volume and neurological deficits. Autophagy and its signaling pathway were determined by evaluating the expression of phosphorylated AMP-activated protein kinase alpha (AMPKα), phosphorylated mammalian target of rapamycin (mTOR), beclin-1, microtubule-associated protein 1A/1B-light chain 3 class II (LC3-II), and p62 by western blotting. Autophagosomes were evaluated by transmission electron microscopy. Neferine treatment significantly reduced infarct volumes and improved neurological deficits. Neferine significantly attenuated the upregulation of autophagy-associated proteins such as LC3-II, beclin-1, and p62 as well as autophagosome formation, all of which were induced by pMCAO. Neferine exerted remarkable protection against cerebral ischemia, possibly via the regulation of autophagy mediated by the Ca2+-dependent AMPK/mTOR pathway.

Associations of ARMS2 and CFH Gene Polymorphisms with Neovascular Age-Related Macular Degeneration.

PURPOSE: This study aimed to determine the association of ARMS2 A69S, ARMS2 del443ins54, and CFH Y402H polymorphisms with neovascular age-related macular degeneration (nAMD) for the first time in an Indonesian population. PATIENTS AND METHODS: Our case-control study involved 104 nAMD and 100 control subjects. AMD diagnosis was evaluated by retinal specialists based on color fundus photography and optical coherence tomography. The polymorphisms on CFH Y402H and ARMS2 A69S were analyzed by PCR-restriction fragment length polymorphism (PCR-RFLP), whereas ARMS2 del443ins54 was evaluated by PCR-based assay. RESULTS: Significant allelic associations with nAMD were detected on all polymorphisms (P<0.05), with stronger association with the ARMS2 A69S (OR 3.13; 95% CI 2.08-4.71; P<0.001) and ARMS2 del443ins54 (OR 3.28; 95% CI 2.17-4.95; P<0.001) polymorphisms than with CFH Y402H (OR 2.08; 95% CI 1.08-3.99; P=0.028). Genotype analysis showed a statistical difference between nAMD and the control group for all polymorphisms (P<0.05). However, the association with nAMD was weaker for CFH Y402H (P=0.043) than for ARMS2 A69S and ARMS2 del443ins54 (P<0.001). A significant interaction between ARMS2 A69S and hypertension was documented (OR 9.53; 95% CI 3.61-25.1; P<0.001). CONCLUSION: Our findings indicate that ARMS2 A69S and ARMS2 del443ins54 polymorphisms are strongly associated with the risk of nAMD for the first time in an Indonesian population. The risk of nAMD increased when the presence of risk alleles from ARMS2 A69S was combined with the presence of hypertension.

Loss of the serine protease HTRA1 impairs smooth muscle cells maturation.

Vascular smooth muscle cell (VSMC) dysfunction is a hallmark of small vessel disease, a common cause of stroke and dementia. Two of the most frequently mutated genes in familial small vessel disease are HTRA1 and NOTCH3. The protease HTRA1 cleaves the NOTCH3 ligand JAG1 implying a mechanistic link between HTRA1 and Notch signaling. Here we report that HTRA1 is essential for VSMC differentiation into the contractile phenotype. Mechanistically, loss of HTRA1 increased JAG1 protein levels and NOTCH3 signaling activity in VSMC. In addition, the loss of HTRA1 enhanced TGFß-SMAD2/3 signaling activity. Activation of either NOTCH3 or TGFß signaling resulted in increased transcription of the HES and HEY transcriptional repressors and promoted the contractile VSMC phenotype. However, their combined over-activation led to an additive accumulation of HES and HEY proteins, which repressed the expression of contractile VSMC marker genes. As a result, VSMC adopted an immature phenotype with impaired arterial vasoconstriction in Htra1-deficient mice. These data demonstrate an essential role of HTRA1 in vascular maturation and homeostasis by controlling Notch and TGFß signaling.

High-Temperature Requirement A1 Protease as a Rate-Limiting Factor in the Development of Osteoarthritis.

Preserving the mature articular cartilage of joints is a critical focus in the prevention and treatment of osteoarthritis. We determined whether the genetic inactivation of high-temperature requirement A1 (HtrA1) can significantly attenuate the degradation of articular or condylar cartilage. Two types of mouse models of osteoarthritis were used, a spontaneous mutant mouse model [type XI collagen-haploinsufficient (Col11a1+/-) mice] and two post-traumatic mouse models [destabilization of the medial meniscus (DMM) on the knee and a partial discectomy (PDE) on the temporomandibular joint]. Three different groups of mice were generated: i) HtrA1 was genetically deleted from Col11a1+/- mice (HtrA1-/-;Col11a1+/-), ii) HtrA1-deficient mice (HtrA1-/-) were subjected to DMM, and iii) HtrA1-/- mice were subjected to PDE. Knee and temporomandibular joints from the mice were characterized for evidence of cartilage degeneration. The degradation of articular or condylar cartilage was significantly delayed in HtrA1-/-;Col11a1+/- mice and HtrA1-/- mice after DMM or PDE. The amount of collagen type VI was significantly higher in the articular cartilage in HtrA1-/-;Col11a1+/- mice, compared with that in Col11a1+/- mice. The genetic removal of HtrA1 may delay the degradation of articular or condylar cartilage in mice.

CADASIL brain vessels show a HTRA1 loss-of-function profile.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and a phenotypically similar recessive condition (CARASIL) have emerged as important genetic model diseases for studying the molecular pathomechanisms of cerebral small vessel disease (SVD). CADASIL, the most frequent and intensely explored monogenic SVD, is characterized by a severe pathology in the cerebral vasculature including the mutation-induced aggregation of the Notch3 extracellular domain (Notch3ECD) and the formation of protein deposits of insufficiently determined composition in vessel walls. To identify key molecules and pathways involved in this process, we quantitatively determined the brain vessel proteome from CADASIL patient and control autopsy samples (n = 6 for each group), obtaining 95 proteins with significantly increased abundance. Intriguingly, high-temperature requirement protein A1 (HTRA1), the extracellular protease mutated in CARASIL, was found to be strongly enriched (4.9-fold, p = 1.6 × 10-3) and to colocalize with Notch3ECD deposits in patient vessels suggesting a sequestration process. Furthermore, the presence of increased levels of several HTRA1 substrates in the CADASIL proteome was compatible with their reduced degradation as consequence of a loss of HTRA1 activity. Indeed, a comparison with the brain vessel proteome of HTRA1 knockout mice (n = 5) revealed a highly significant overlap of 18 enriched proteins (p = 2.2 × 10-16), primarily representing secreted and extracellular matrix factors. Several of them were shown to be processed by HTRA1 in an in vitro proteolysis assay identifying them as novel substrates. Our study provides evidence for a loss of HTRA1 function as a critical step in the development of CADASIL pathology linking the molecular mechanisms of two distinct SVD forms.

Loss of HtrA1 serine protease induces synthetic modulation of aortic vascular smooth muscle cells.

Homozygous mutations of human HTRA1 cause cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). HtrA1-/- mice were examined for arterial abnormalities. Although their cerebral arteries were normal, the thoracic aorta was affected in HtrA1-/- mice. The number of vascular smooth muscle cells (VSMCs) in the aorta was increased in HtrA1-/- mice of 40 weeks or younger, but decreased thereafter. The cross-sectional area of the aorta was increased in HtrA1-/- mice of 40 weeks or older. Aortic VSMCs isolated from HtrA1-/- mice rapidly proliferated and migrated, produced high MMP9 activity, and were prone to oxidative stress-induced cell death. HtrA1-/- VSMCs expressed less smooth muscle α-actin, and more vimentin and osteopontin, and responded to PDGF-BB more strongly than wild type VSMCs, indicating that HtrA1-/- VSMCs were in the synthetic phenotype. The elastic lamina was disrupted, and collagens were decreased in the aortic media. Calponin in the media was decreased, whereas vimentin and osteopontin were increased, suggesting a synthetic shift of VSMCs in vivo. Loss of HtrA1 therefore skews VSMCs toward the synthetic phenotype, induces MMP9 expression, and expedites cell death. We propose that the synthetic modulation is the primary event that leads to the vascular abnormalities caused by HtrA1 deficiency.

Inactivation of the serine protease HTRA1 inhibits tumor growth by deregulating angiogenesis.

The serine protease HTRA1 is involved in several vascular diseases and its expression is often deregulated in cancer. We aimed at identifying how HTRA1 in the vasculature affects tumor growth. Here we report that silencing of HTRA1 in cultured endothelial cells increased migration rate and tube formation, whereas forced HTRA1 expression impaired sprouting angiogenesis. Mechanistically, endothelial HTRA1 expression enhanced Delta/Notch signaling by reducing the amount of the weak Notch ligand JAG1. HTRA1 physically interacted with JAG1 and cleaved it within the intracellular domain, leading to protein degradation. Expression of a constitutive active Notch1 prevented the hypersprouting phenotype upon silencing of HTRA1. In HtrA1-deficient mice, endothelial Notch signaling was diminished and isolated endothelial cells had increased expression of VEGF receptor-2. Growth of syngeneic tumors was strongly impaired in HtrA1-/- mice. The tumor vasculature was much denser in HtrA1-/- mice and less covered with mural cells. This chaotic and immature vascular network was poorly functional as indicated by large hypoxic tumor areas and low tumor cell proliferation rates. In summary, inhibition of HTRA1 in the tumor stroma impaired tumor progression by deregulating angiogenesis.

BMP-Responsive Protease HtrA1 Is Differentially Expressed in Astrocytes and Regulates Astrocytic Development and Injury Response.

Astrocytes perform a wide array of physiological functions, including structural support, ion exchange, and neurotransmitter uptake. Despite this diversity, molecular markers that label subpopulations of astrocytes are limited, and mechanisms that generate distinct astrocyte subtypes remain unclear. Here we identified serine protease high temperature requirement A 1 (HtrA1), a bone morphogenetic protein 4 signaling regulated protein, as a novel marker of forebrain astrocytes, but not of neural stem cells, in adult mice of both sexes. Genetic deletion of HtrA1 during gliogenesis accelerates astrocyte differentiation. In addition, ablation of HtrA1 in cultured astrocytes leads to altered chondroitin sulfate proteoglycan expression and inhibition of neurite extension, along with elevated levels of transforming growth factor-ß family proteins. Brain injury induces HtrA1 expression in reactive astrocytes, and loss of HtrA1 leads to an impairment in wound closure accompanied by increased proliferation of endothelial and immune cells. Our findings demonstrate that HtrA1 is differentially expressed in adult mouse forebrain astrocytes, and that HtrA1 plays important roles in astrocytic development and injury response.SIGNIFICANCE STATEMENT Astrocytes, an abundant cell type in the brain, perform a wide array of physiological functions. Although characterized as morphologically and functionally diverse, molecular markers that label astrocyte subtypes or signaling pathways that lead to their diversity remain limited. Here, after examining the expression profile of astrocytes generated in response to bone morphogenetic protein signaling, we identify high temperature requirement A 1 (HtrA1) as an astrocyte-specific marker that is differentially expressed in distinct adult mouse brain regions. HtrA1 is a serine protease that has been linked to cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy, a small blood vessel disease in humans. Understanding the role of HtrA1 during development and after injury will provide insights into how distinct astrocyte populations are generated and their unique roles in injury and disease.

Role of HTRA1 in bone formation and regeneration: In vitro and in vivo evaluation.

The role of mammalian high temperature requirement protease A1 (HTRA1) in somatic stem cell differentiation and mineralized matrix formation remains controversial, having been demonstrated to impart either anti- or pro-osteogenic effects, depending on the in vitro cell model used. The aim of this study was therefore to further evaluate the role of HTRA1 in regulating the differentiation potential and lineage commitment of murine mesenchymal stem cells in vitro, and to assess its influence on bone structure and regeneration in vivo. Our results demonstrated that short hairpin RNA-mediated ablation of Htra1 in the murine mesenchymal cell line C3H10T1/2 increased the expression of several osteogenic gene markers, and significantly enhanced matrix mineralization in response to BMP-2 stimulation. These effects were concomitant with decreases in the expression of chondrogenic gene markers, and increases in adipogenic gene expression and lipid accrual. Despite the profound effects of loss-of-function of HTRA1 on this in vitro osteochondral model, these were not reproduced in vivo, where bone microarchitecture and regeneration in 16-week-old Htra1-knockout mice remained unaltered as compared to wild-type controls. By comparison, analysis of femurs from 52-week-old mice revealed that bone structure was better preserved in Htra1-knockout mice than age-matched wild-type controls. These findings therefore provide additional insights into the role played by HTRA1 in regulating mesenchymal stem cell differentiation, and offer opportunities for improving our understanding of how this multifunctional protease may act to influence bone quality.

Epigenetic silencing of serine protease HTRA1 drives polyploidy.

BACKGROUND: Increased numbers and improperly positioned centrosomes, aneuploidy or polyploidy, and chromosomal instability are frequently observed characteristics of cancer cells. While some aspects of these events and the checkpoint mechanisms are well studied, not all players have yet been identified. As the role of proteases other than the proteasome in tumorigenesis is an insufficiently addressed question, we investigated the epigenetic control of the widely conserved protease HTRA1 and the phenotypes of deregulation. METHODS: Mouse embryonal fibroblasts and HCT116 and SW480 cells were used to study the mechanism of epigenetic silencing of HTRA1. In addition, using cell biological and genetic methods, the phenotypes of downregulation of HTRA1 expression were investigated. RESULTS: HTRA1 is epigenetically silenced in HCT116 colon carcinoma cells via the epigenetic adaptor protein MBD2. On the cellular level, HTRA1 depletion causes multiple phenotypes including acceleration of cell growth, centrosome amplification and polyploidy in SW480 colon adenocarcinoma cells as well as in primary mouse embryonic fibroblasts (MEFs). CONCLUSIONS: Downregulation of HTRA1 causes a number of phenotypes that are hallmarks of cancer cells suggesting that the methylation state of the HtrA1 promoter may be used as a biomarker for tumour cells or cells at risk of transformation.

Synthesis, Photophysical Properties, and Biological Evaluation of trans-Bisthioglycosylated Tetrakis(fluorophenyl)chlorin for Photodynamic Therapy.

trans-Bisthioglycosylated tetrakis(fluorophenyl)chlorin (7) was designed as a powerful photodynamic therapy (PDT) photosensitizer based on the findings of our systematic studies. We show here that the trans-bisthioglycosylated structure of 7 enhanced its uptake by HeLa cells and that the chlorin ring of 7 increased the efficiency of reactive oxygen species generation under the standard condition of our photocytotoxicity test. The versatility of 7 in PDT treatment was established using weakly metastatic B16F1 melanoma cells, metastatic 4T1 breast cancer cells, the RGK-1 gastric carcinoma mucosal cell line, and three human glioblastoma cell lines (U87, U251, and T98G). The pharmacokinetics of 7 in mice bearing 4T1 breast cancer cells showed a high tumor-to-skin concentration ratio (approximately 60) at 24 h after intraperitoneal injection. The PDT efficacy of 7 in vivo was approximately 250-times higher than that of mono-l-aspartyl chlorin e6 (9) in mice bearing 4T1 breast cancer cells.