Identification of highly potent and selective HTRA1 inhibitors.

High temperature requirement A serine peptidase 1 (HTRA1) is a serine protease involved in an array of signaling pathways. It is also responsible for the regulation of protein aggregates via refolding, translocation, and degradation. It has subsequently been found that runaway proteolytic HTRA1 activity plays a role in a variety of diseases, including Age-Related Macular Degeneration (AMD), osteoarthritis, and Rheumatoid Arthritis. Selective inhibition of serine protease HTRA1 therefore offers a promising new strategy for the treatment of these diseases. Herein we disclose structure-activity-relationship (SAR) studies which identify key interactions responsible for binding affinity of small molecule inhibitors to HTRA1. The study results in highly potent molecules with IC50's less than 15 nM and excellent selectivity following a screen of 35 proteases.

Heterogeneous blood-brain barrier dysfunction in cerebral small vessel diseases.

INTRODUCTION: We explored how blood-brain barrier (BBB) leakage rate of gadolinium chelates (Ktrans) and BBB water exchange rate (kw) varied in cerebral small vessel disease (cSVD) subtypes. METHODS: Thirty sporadic cSVD, 40 cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), and 13 high-temperature requirement factor A serine peptidase 1 (HTRA) -related cSVD subjects were investigated parallel to 40 healthy individuals. Subjects underwent clinical, cognitive, and MRI assessment. RESULTS: In CADASIL, no difference in Ktrans, but lower kw was observed in multiple brain regions. In sporadic cSVD, no difference in kw, but higher Ktrans was found in the whole brain and normal-appearing white matter. In HTRA1-related cSVD, both higher Ktrans in the whole brain and lower kw in multiple brain regions were observed. In each patient group, the altered BBB measures were correlated with lesion burden or clinical severity. DISCUSSION: In cSVD subtypes, distinct alterations of kw and Ktrans were observed. The combination of Ktrans and kw can depict the heterogeneous BBB dysfunction. HIGHLIGHTS: We measured BBB leakage to gadolinium-based contrast agent (Ktrans) and water exchange rate (kw) across BBB in three subtypes of cSVD. CADASIL is characterized by lower kw, HTRA1-related cSVD exhibits both higher Ktrans and lower kw, while sporadic cSVD is distinguished by higher Ktrans. There are distinct alterations in kw and Ktrans among subtypes of cSVD, indicating the heterogeneous nature of BBB dysfunction.

Progress in the Study of the Role and Mechanism of HTRA1 in Diseases Related to Vascular Abnormalities.

High temperature requirement A1 (HTRA1) is a member of the serine protease family, comprising four structural domains: IGFBP domain, Kazal domain, protease domain and PDZ domain. HTRA1 encodes a serine protease, a secreted protein that is widely expressed in the vasculature. HTRA1 regulates a wide range of physiological processes through its proteolytic activity, and is also involved in a variety of vascular abnormalities-related diseases. This article reviews the role of HTRA1 in the development of vascular abnormalities-related hereditary cerebral small vessel disease (CSVD), age-related macular degeneration (AMD), tumors and other diseases. Through relevant research advances to understand the role of HTRA1 in regulating signaling pathways or refolding, translocation, degradation of extracellular matrix (ECM) proteins, thus directly or indirectly regulating angiogenesis, vascular remodeling, and playing an important role in vascular homeostasis, further understanding the mechanism of HTRA1's role in vascular abnormality-related diseases is important for HTRA1 to be used as a therapeutic target in related diseases.

HTRA1-driven detachment of type I collagen from endoplasmic reticulum contributes to myocardial fibrosis in dilated cardiomyopathy.

BACKGROUND: The aberrant secretion and excessive deposition of type I collagen (Col1) are important factors in the pathogenesis of myocardial fibrosis in dilated cardiomyopathy (DCM). However, the precise molecular mechanisms underlying the synthesis and secretion of Col1 remain unclear. METHODS AND RESULTS: RNA-sequencing analysis revealed an increased HtrA serine peptidase 1 (HTRA1) expression in patients with DCM, which is strongly correlated with myocardial fibrosis. Consistent findings were observed in both human and mouse tissues by immunoblotting, quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunohistochemistry, and immunofluorescence analyses. Pearson's analysis showed a markedly positive correlation between HTRA1 level and myocardial fibrosis indicators, including extracellular volume fraction (ECV), native T1, and late gadolinium enhancement (LGE), in patients with DCM. In vitro experiments showed that the suppression of HTRA1 inhibited the conversion of cardiac fibroblasts into myofibroblasts and decreased Col1 secretion. Further investigations identified the role of HTRA1 in promoting the formation of endoplasmic reticulum (ER) exit sites, which facilitated the transportation of Col1 from the ER to the Golgi apparatus, thereby increasing its secretion. Conversely, HTRA1 knockdown impeded the retention of Col1 in the ER, triggering ER stress and subsequent induction of ER autophagy to degrade misfolded Col1 and maintain ER homeostasis. In vivo experiments using adeno-associated virus-serotype 9-shHTRA1-green fluorescent protein (AAV9-shHTRA1-GFP) showed that HTRA1 knockdown effectively suppressed myocardial fibrosis and improved left ventricular function in mice with DCM. CONCLUSIONS: The findings of this study provide valuable insights regarding the treatment of DCM-associated myocardial fibrosis and highlight the therapeutic potential of targeting HTRA1-mediated collagen secretion.

HTRA1 promotes EMT through the HDAC6/Ac-α-tubulin pathway in human GBM cells.

BACKGROUND: The infiltrative nature of human gliomas renders complete surgical removal of tumors futile. Thus, illuminating mechanisms of their infiltrative properties may improve therapies and outcomes of glioma patients. METHODS: Comprehensive bioinformatic analyses of PRSS family were undertaken. Transfection of HTRA1 siRNAs was used to suppress HTRA1 expression. CCK-8, EdU, and colony formation assay were employed to assess cell viability, and cell migration/invasion was detected by transwell, wound healing, and 3D tumor spheroid invasion assays. Immunoprecipitation was applied to study the mechanism that HTRA1 affected cell migration. In addition, in situ xenograft tumor model was employed to explore the role of HTRA1 in glioma growth in vivo. RESULTS: HTRA1 knockdown could lead to suppression of cell viability, migration and invasion, as well as increased apoptosis. Immunoprecipitation results indicates HTRA1 might facilitate combination between HDAC6 and α-tubulin to enhance cell migration by decreasing α-tubulin acetylation. Besides, HTRA1 knockdown inhibited the growth of xenografts derived from orthotopic implantation of GBM cells and prolonged the survival time of tumor-bearing mice. CONCLUSION: Our results indicate that HTRA1 promotes the proliferation and migration of GBM cells in vitro and in vivo, and thus may be a potential target for treatment in gliomas.

Possible involvement of HtrA1 serine protease in the onset of osteoporotic bone extracellular matrix changes.

High-temperature requirement A1 (HtrA1), a multidomain serine protease acting on Extracellular matrix (ECM) rearrangement, is also secreted by osteoblasts and osteoclasts. Recent and conflicting literature highlights HtrA1's role as a controller of bone remodeling, proposing it as a possible target for pathologies with unbalanced bone resorption, like Osteoporosis (OP). To add knowledge on this molecule function in bone physiopathology, here we compared HtrA1 distribution in the ECM of healthy (H) and OP bone tissue, also examining its localization in the sites of new bone formation. HtrA1 was homogeneously expressed in the mature bone ECM of H tissue showing a 55.6 ± 16.4% of the stained area, with a significant (p=0.0001) decrease in OP percentage stained area (21.1 ± 13.1). Moreover, HtrA1 was present in the endosteum and cells involved in osteogenesis, mainly in those "entrapped" in woven bone, whereas osteocytes in mature lamellar bone were negative. Based on our previous observation in OP tissue of a significantly increased expression of Decorin and Osteocalcin, both involved in bone mineralization and remodeling and equally substrates for HtrA1, we speculate that HtrA1 by controlling the proper amount of Decorin and Osteocalcin favors normal bone maturation and mineralization. Besides, we suggest that late-osteoblasts and pre-osteocytes secrete HtrA1 in the adjacent matrix whilst proceeding with their maturation and that HtrA1 expression is further modified during the remodeling from woven to the lamellar bone. Overall, our data suggest HtrA1 as a positive regulator of bone matrix formation and maturation: its reduced expression in mature OP bone, affecting protein content and distribution, could hamper correct bone remodeling and mineralization.

Systematic Characterization of the Clinical and Pathological Features of Schwannomas Harboring SH3PXD2A::HTRA1 Fusion.

The understanding of schwannoma tumorigenesis has been reshaped by the recent identification of SH3PXD2A::HTRA1 fusion in 10% of intracranial/spinal schwannomas. Nonetheless, pathologic features of schwannomas harboring this fusion, as well as its prevalence outside intracranial/spinal locations, have not been characterized. We screened 215 consecutive schwannomas for their clinicopathologic characteristics and fusion status using reverse-transcriptase polymerase chain reaction (RT-PCR). Among 29 (13.5%) fusion-positive schwannomas, the most prevalent location was peripheral somatic tissue (30.7%, 19/62), followed by spinal/paraspinal (18.4%, 7/38), body cavity/deep structures (10%, 2/20), intracranial (1.3%, 1/75), and viscera (0/13). All 8 cellular, 4 microcystic/reticular, and 3 epithelioid schwannomas were fusion-negative, as were 41/42 nonschwannomatous peripheral nerve sheath tumors. Remarkably, a distinct 'serpentine' palisading pattern, comprising ovoid/plump cells shorter than usual schwannian cells in a hyalinized stroma, was identified in most fusion-positive cases and the schwannomatous component of the only fusion-positive malignant peripheral nerve sheath tumor. To validate this finding, 60 additional cases were collected, including 36 with (≥10% arbitrarily) and 24 without appreciable serpentine histology, of which 29 (80.6%) and 2 (8.3%) harbored the fusion, respectively. With percentages of 'serpentine' areas scored, 10% was determined as the optimal practical cut-off to predict the fusion status (sensitivity, 0.950; specificity, 0.943). Fusion positivity was significantly associated with serpentine histology, smaller tumors, younger patients, and peripheral somatic tissue, while multivariate logistic linear regression analysis only identified serpentine histology and location as independent fusion-predicting factors. RNA in situ hybridization successfully detected the fusion junction, highly concordant with RT-PCR results. Gene expression profiling on 18 schwannomas demonstrated segregation largely consistent with fusion status. Fusion-positive cases expressed significantly higher HTRA1 mRNA abundance, perhaps exploitable as a biomarker. In summary, we systematically characterize a series of 60 SH3PXD2A::HTRA1 fusion-positive schwannomas, showing their distinctive morphology and location-specific prevalence for the first time.

High temperature requirement A1 and macrophage migration inhibitory factor in the cerebrospinal fluid; a potential marker of conversion from relapsing-remitting to secondary progressive multiple sclerosis.

BACKGROUND: Predictive and prognostic biomarkers for multiple sclerosis (MS) remain a significant gap in MS diagnosis and treatment monitoring. Currently, there are no timely markers to diagnose the transition to secondary progressive MS (SPMS). OBJECTIVE: This study aims to evaluate the discriminatory potential of the High temperature requirement serine protease (HTRA1)/Macrophage migration inhibitory factor (MIF) cerebrospinal fluid (CSF) ratio in distinguishing relapsing-remitting (RRMS) patients from SPMS patients. METHODS: The MIF and HTRA1 CSF levels were determined using ELISA in healthy controls (n = 23), RRMS patients before (n = 22) and after 1 year of dimethyl fumarate treatment (n = 11), as well as in SPMS patients before (n = 11) and after 2 years of mitoxantrone treatment (n = 7). The ability of the HTRA1/MIF ratio to discriminate the different groups was determined using receiver operating curve (ROC) analyses. RESULTS: The ratio was significantly increased in treatment naïve RRMS patients while decreased again in SPMS patients at baseline. Systemic administrated disease modifying treatment (DMT) only significantly affected the ratio in RRMS patients. ROC analysis demonstrated that the ratio could discriminate treatment naïve RRMS patients from SPMS patients with 91% sensitivity and 100% specificity. CONCLUSION: The HTRA1/MIF ratio is a strong candidate as a MS biomarker for SPMS conversion.

Critical Dependence on Area in Relationship between ARMS2/HTRA1 Genotype and Faster Geographic Atrophy Enlargement: Age-Related Eye Disease Study 2 Report Number 33.

PURPOSE: To analyze ARMS2/HTRA1 as a risk factor for faster geographic atrophy (GA) enlargement according to (1) GA area and (2) contiguous enlargement versus progression to multifocality. DESIGN: Age-Related Eye Disease Study 2 (AREDS2) cohort analysis. PARTICIPANTS: Eyes with GA: 546 eyes of 406 participants. METHODS: Geographic atrophy area was measured from color fundus photographs at annual visits. Mixed-model regression of square root of GA area and proportional hazards regression of progression to multifocality were analyzed by ARMS2 genotype. MAIN OUTCOME MEASURES: Change in square root GA area and progression to multifocality. RESULTS: Geographic atrophy enlargement was significantly faster with ARMS2 risk alleles (P < 0.0001) at 0.224 mm/year (95% CI, 0.195-0.252 mm/year), 0.298 mm/year (95% CI, 0.271-0.324 mm/year), and 0.317 mm/year (95% CI, 0.279-0.355 mm/year), for 0 to 2 risk alleles, respectively. However, a significant interaction (P = 0.011) was observed between genotype and baseline area. In eyes with very small area (< 1.9 mm2), enlargement was significantly faster with ARMS2 risk alleles (P < 0.0001) at 0.193 mm/year (95% CI, 0.162-0.225 mm/year) versus 0.304 mm/year (95% CI, 0.280-0.329 mm/year) for 0 versus 1 to 2 risk alleles, respectively. With moderately small (1.9-3.8 mm2) or medium to large (≥ 3.8 mm2) area, enlargement was not significantly faster with ARMS2 risk alleles (P = 0.66 and P = 0.70, respectively). In nonmultifocal GA, enlargement was significantly faster with ARMS2 risk alleles (P = 0.001) at 0.175 mm/year (95% CI, 0.142-0.209 mm/year), 0.226 mm/year (95% CI, 0.193-0.259 mm/year), and 0.287 mm/year (95% CI, 0.237-0.337 mm/year) with 0 to 2 risk alleles, respectively. ARMS2 genotype was not associated significantly with progression to multifocal GA. CONCLUSIONS: The relationship between ARMS2/HTRA1 genotype and faster GA enlargement depends critically on GA area: risk alleles represent a strong risk factor for faster enlargement only in eyes with very small area. They increase the growth rate more through contiguous enlargement than progression to multifocality. ARMS2/HTRA1 genotype is more important in increasing risk of progression to GA and initial GA enlargement (contiguously) than in subsequent enlargement or progression to multifocality. These findings may explain some discrepancies between previous studies and have implications for both research and clinical practice. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Identification of osteoarthritis-characteristic genes and immunological micro-environment features through bioinformatics and machine learning-based approaches.

BACKGROUND: Osteoarthritis (OA) is a multifaceted chronic joint disease characterized by complex mechanisms. It has a detrimental impact on the quality of life for individuals in the middle-aged and elderly population while also imposing a significant socioeconomic burden. At present, there remains a lack of comprehensive understanding regarding the pathophysiology of OA. The objective of this study was to examine the genes, functional pathways, and immune infiltration characteristics associated with the development and advancement of OA. METHODS: The Gene Expression Omnibus (GEO) database was utilized to acquire gene expression profiles. The R software was employed to conduct the screening of differentially expressed genes (DEGs) and perform enrichment analysis on these genes. The OA-characteristic genes were identified using the Weighted Gene Co-expression Network Analysis (WGCNA) and the Lasso algorithm. In addition, the infiltration levels of immune cells in cartilage were assessed using single-sample gene set enrichment analysis (ssGSEA). Subsequently, a correlation analysis was conducted to examine the relationship between immune cells and the OA-characteristic genes. RESULTS: A total of 80 DEGs were identified. As determined by functional enrichment, these DEGs were associated with chondrocyte metabolism, apoptosis, and inflammation. Three OA-characteristic genes were identified using WGCNA and the lasso algorithm, and their expression levels were then validated using the verification set. Finally, the analysis of immune cell infiltration revealed that T cells and B cells were primarily associated with OA. In addition, Tspan2, HtrA1 demonstrated a correlation with some of the infiltrating immune cells. CONCLUSIONS: The findings of an extensive bioinformatics analysis revealed that OA is correlated with a variety of distinct genes, functional pathways, and processes involving immune cell infiltration. The present study has successfully identified characteristic genes and functional pathways that hold potential as biomarkers for guiding drug treatment and facilitating molecular-level research on OA.

Current Views on Chr10q26 Contribution to Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness in the global aging population. Familial aggregation and genome-wide association (GWA) studies have identified gene variants associated with AMD, implying a strong genetic contribution to AMD development. Two loci, on human Chr 1q31 and 10q26, respectively, represent the most influential of all genetic factors. While the role of CFH at Chr 1q31 is well established, uncertainty remains about the genes ARMS2 and HTRA1, at the Chr 10q26 locus. Since both genes are in strong linkage disequilibrium, assigning individual gene effects is difficult. In this chapter, we review current literature about ARMS2 and HTRA1 and their relevance to AMD risk. Future studies will be necessary to unravel the mechanisms by which they contribute to AMD.

HTRA1 from OVX rat osteoclasts causes detrimental effects on endplate chondrocytes through NF-κB.

Endplate osteochondritis is considered one of the major causes of intervertebral disc degeneration (IVDD) and low back pain. Menopausal women have a higher rate of endplate cartilage degeneration than similarly aged men, but the related mechanisms are still unclear. Subchondral bone changes, mainly mediated by osteoblasts and osteoclasts, are considered an important reason for the degeneration of cartilage. This work explored the role of osteoclasts in endplate cartilage degeneration, as well as its underlying mechanisms. A rat ovariectomy (OVX) model was used to induce estrogen deficiency. Our experiments indicated that OVX significantly promoted osteoclastogenesis and anabolism and catabolism changes in endplate chondrocytes. OVX osteoclasts cause an imbalance between anabolism and catabolism in endplate chondrocytes, as shown by a decrease in anabolic markers such as Aggrecan and Collagen II, and an increase in catabolic markers such as a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and matrix metalloproteinases (MMP13). Osteoclasts were also confirmed in this study to be able to secrete HtrA serine peptidase 1 (HTRA1), which resulted in increased catabolism in endplate chondrocytes through the NF-κB pathway under estrogen deficiency. This study demonstrated the involvement and mechanism of osteoclasts in the anabolism and catabolism changes of endplate cartilage under estrogen deficiency, and proposed a new strategy for the treatment of endplate osteochondritis and IVDD by targeting HTRA1.

Rare neurovascular genetic and imaging markers across neurodegenerative diseases.

INTRODUCTION: Cerebral small vessel disease (SVD) is common in patients with cognitive impairment and neurodegenerative diseases such as Alzheimer's and Parkinson's. This study investigated the burden of magnetic resonance imaging (MRI)-based markers of SVD in patients with neurodegenerative diseases as a function of rare genetic variant carrier status. METHODS: The Ontario Neurodegenerative Disease Research Initiative study included 520 participants, recruited from 14 tertiary care centers, diagnosed with various neurodegenerative diseases and determined the carrier status of rare non-synonymous variants in five genes (ABCC6, COL4A1/COL4A2, NOTCH3/HTRA1). RESULTS: NOTCH3/HTRA1 were found to significantly influence SVD neuroimaging outcomes; however, the mechanisms by which these variants contribute to disease progression or worsen clinical correlates are not yet understood. DISCUSSION: Further studies are needed to develop genetic and imaging neurovascular markers to enhance our understanding of their potential contribution to neurodegenerative diseases.

Inhibiting HIF-1 signaling alleviates HTRA1-induced RPE senescence in retinal degeneration.

BACKGROUND: Age-related macular degeneration (AMD), characterized by the degeneration of retinal pigment epithelium (RPE) and photoreceptors, is the leading cause of irreversible vision impairment among the elderly. RPE senescence is an important contributor to AMD and has become a potential target for AMD therapy. HTRA1 is one of the most significant susceptibility genes in AMD, however, the correlation between HTRA1 and RPE senescence hasn't been investigated in the pathogenesis of AMD. METHODS: Western blotting and immunohistochemistry were used to detect HTRA1 expression in WT and transgenic mice overexpressing human HTRA1 (hHTRA1-Tg mice). RT-qPCR was used to detect the SASP in hHTRA1-Tg mice and ARPE-19 cells infected with HTRA1. TEM, SA-ß-gal was used to detect the mitochondria and senescence in RPE. Retinal degeneration of mice was investigated by fundus photography, FFA, SD-OCT and ERG. The RNA-Seq dataset of ARPE-19 cells treated with adv-HTRA1 versus adv-NC were analyzed. Mitochondrial respiration and glycolytic capacity in ARPE-19 cells were measured using OCR and ECAR. Hypoxia of ARPE-19 cells was detected using EF5 Hypoxia Detection Kit. KC7F2 was used to reduce the HIF1α expression both in vitro and in vivo. RESULTS: In our study, we found that RPE senescence was facilitated in hHTRA1-Tg mice. And hHTRA1-Tg mice became more susceptible to NaIO3 in the development of oxidative stress-induced retinal degeneration. Similarly, overexpression of HTRA1 in ARPE-19 cells accelerated cellular senescence. Our RNA-seq revealed an overlap between HTRA1-induced differentially expressed genes associated with aging and those involved in mitochondrial function and hypoxia response in ARPE-19 cells. HTRA1 overexpression in ARPE-19 cells impaired mitochondrial function and augmented glycolytic capacity. Importantly, upregulation of HTRA1 remarkably activated HIF-1 signaling, shown as promoting HIF1α expression which mainly located in the nucleus. HIF1α translation inhibitor KC7F2 significantly prevented HTRA1-induced cellular senescence in ARPE-19 cells, as well as improved the visual function in hHTRA1-Tg mice treated with NaIO3. CONCLUSIONS: Our study showed elevated HTRA1 contributes to the pathogenesis of AMD by promoting cellular senescence in RPE through damaging mitochondrial function and activating HIF-1 signaling. It also pointed out that inhibition of HIF-1 signaling might serve as a potential therapeutic strategy for AMD. Video Abstract.

Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL): A challenging diagnosis and a rare multiple sclerosis mimic.

Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is an extremely rare hereditary cerebral small vessel disease caused by homozygous or compound heterozygous mutations in the gene coding for high-temperature requirement A serine peptidase 1 (HtrA1). Given the rare nature of the disease, delays in diagnosis and misdiagnosis are not uncommon. In this article, we reported the first case of CARASIL from Saudi Arabia with a novel homozygous variant c.1156C>T in exon 7 of the HTRA1 gene. The patient was initially misdiagnosed with primary progressive multiple sclerosis and treated with rituximab. CARASIL should be considered in the differential diagnosis of patients with suspected atypical progressive multiple sclerosis who have additional signs such as premature scalp alopecia and low back pain with diffuse white matter lesions in brain MRI. Genetic testing is important to confirm the diagnosis.

HTRA1 in Placental Cell Models: A Possible Role in Preeclampsia.

The HtrA serine peptidase 1 (HTRA1) is a multidomain secretory protein with serine-protease activity involved in the regulation of many cellular processes in both physiological and pathological conditions. HTRA1 is normally expressed in the human placenta, and its expression is higher in the first trimester compared to the third trimester, suggesting an important role of this serine protease in the early phases of human placenta development. The aim of this study was to evaluate the functional role of HTRA1 in in vitro models of human placenta in order to define the role of this serine protease in preeclampsia (PE). BeWo and HTR8/SVneo cells expressing HTRA1 were used as syncytiotrophoblast and cytotrophoblast models, respectively. Oxidative stress was induced by treating BeWo and HTR8/SVneo cells with H2O2 to mimic PE conditions in order to evaluate its effect on HTRA1 expression. In addition, HTRA1 overexpression and silencing experiments were performed to evaluate the effects on syncytialization, cell mobility, and invasion processes. Our main data showed that oxidative stress significantly increased HTRA1 expression in both BeWo and HTR8/SVneo cells. In addition, we demonstrated that HTRA1 has a pivotal role in cell motility and invasion processes. In particular, HTRA1 overexpression increased while HTRA1 silencing decreased cell motility and invasion in HTR8/SVneo cell model. In conclusion, our results suggest an important role of HTRA1 in regulating extravillous cytotrophoblast invasion and motility during the early stage of placentation in the first trimester of gestation, suggesting a key role of this serine protease in PE onset.

A human induced pluripotent stem cell model from a patient with hereditary cerebral small vessel disease carrying a heterozygous R302Q mutation in HTRA1.

Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is an inherited cerebral small vessel disease (CSVD) caused by biallelic mutations in the high-temperature requirement serine peptidase A1 (HTRA1) gene. Even heterozygous mutations in HTRA1 are recently revealed to cause cardinal clinical features of CSVD. Here, we report the first establishment of a human induced pluripotent stem cell (hiPSC) line from a patient with heterozygous HTRA1-related CSVD. Peripheral blood mononuclear cells (PBMCs) were reprogrammed by the transfection of episomal vectors encoding human OCT3/4 (POU5F1), SOX2, KLF4, L-MYC, LIN28, and a murine dominant-negative mutant of p53 (mp53DD). The established iPSCs had normal morphology as human pluripotent stem cells and normal karyotype (46XX). Moreover, we found that the HTRA1 missense mutation (c.905G>A, p.R302Q) was heterozygous. These iPSCs expressed pluripotency-related markers and had the potential to differentiate into all three germ layers in vitro. HTRA1 and the supposed disease-associated gene NOG were differentially expressed in the patient iPSCs at mRNA levels compared to those of control lines. The iPSC line would facilitate in vitro research for understanding the cellular pathomechanisms caused by the HTRA1 mutation including its dominant-negative effect.

Heterozygous Pathogenic and Likely Pathogenic Symptomatic HTRA1 Variant Carriers in Cerebral Small Vessel Disease.

High temperature requirement serine peptidase A1 (HTRA1) related cerebral small vessel disease (CSVD) includes both symptomatic heterozygous HTRA1 variant carrier and cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) patients. Presently, most reported symptomatic heterozygous HTRA1 variant carrier cases are sporadic family reports with a lack of specific characteristics. Additionally, the molecular mechanism of heterozygous HTRA1 gene variants is unclear. We conducted this review to collect symptomatic carriers of heterozygous HTRA1 gene variants reported as of 2022, analyzed all pathogenicity according to American College of Medical Genetics and Genomics (ACMG) variant classification, and summarized the cases with pathogenic and likely pathogenic HTRA1 variants gender characteristics, age of onset, geographical distribution, initial symptoms, clinical manifestations, imaging signs, HTRA1 gene variant information and to speculate its underlying pathogenic mechanisms. In this review, we summarized the following characteristics of pathogenic and likely pathogenic symptomatic HTRA1 variant carriers: to date, the majority of reported symptomatic HTRA1 carriers are in European and Asian countries, particularly in China which was found to have the highest number of reported cases. The age of first onset is mostly concentrated in the fourth and fifth decades. The heterozygous HTRA1 gene variants were mostly missense variants. The two variant sites, 166-182 aa and 274-302 aa, were the most concentrated. Clinicians need to pay attention to de novo data and functional data, which may affect the pathogenicity analysis. The decrease in HtrA1 protease activity is currently the most important explanation for the genetic pathogenesis.

Genome-wide association study and identification of systemic comorbidities in development of age-related macular degeneration in a hospital-based cohort of Han Chinese.

Background: Age-related macular degeneration (AMD) is the main cause of severe vision loss in elderly populations of the developed world with limited therapeutic medications available. It is a multifactorial disease with a strong genetic susceptibility which exhibits the differential genetic landscapes among different ethnic groups. Methods: To investigate the Han Chinese-specific genetic variants for AMD development and progression, we have presented a genome-wide association study (GWAS) on 339 AMD cases and 3,390 controls of a Han Chinese population recruited from the Taiwan Precision Medicine Initiative (TPMI). Results: In this study, we have identified several single nucleotide polymorphisms (SNPs) significantly associated with AMD, including rs10490924, rs3750848, and rs3750846 in the ARMS2 gene, and rs3793917, rs11200638, and rs2284665 in the HTRA1 gene, in which rs10490924 was highly linked to the other variants based upon linkage disequilibrium analysis. Moreover, certain systemic comorbidities, including chronic respiratory diseases and cerebrovascular diseases, were also confirmed to be independently associated with AMD. Stratified analysis revealed that both non-exudative and exudative AMD were significantly correlated with these risk factors. We also found that homozygous alternate alleles of rs10490924 could lead to an increased risk of AMD incidence compared to homozygous references or heterozygous alleles in the cohorts of chronic respiratory disease, cerebrovascular disease, hypertension, and hyperlipidemia. Ultimately, we established the SNP models for AMD risk prediction and found that rs10490924 combined with the other AMD-associated SNPs identified from GWAS improved the prediction model performance. Conclusion: These results suggest that genetic variants combined with the comorbidities could effectively identify any potential individuals at a high risk of AMD, thus allowing for both early prevention and treatment.