Sox9 Accelerates Vascular Aging by Regulating Extracellular Matrix Composition and Stiffness.

BACKGROUND: Vascular calcification and increased extracellular matrix (ECM) stiffness are hallmarks of vascular aging. Sox9 (SRY-box transcription factor 9) has been implicated in vascular smooth muscle cell (VSMC) osteo/chondrogenic conversion; however, its relationship with aging and calcification has not been studied. METHODS: Immunohistochemistry was performed on human aortic samples from young and aged patients. Young and senescent primary human VSMCs were induced to produce ECM, and Sox9 expression was manipulated using adenoviral overexpression and depletion. ECM properties were characterized using atomic force microscopy and proteomics, and VSMC phenotype on hydrogels and the ECM were examined using confocal microscopy. RESULTS: In vivo, Sox9 was not spatially associated with vascular calcification but correlated with the senescence marker p16 (cyclin-dependent kinase inhibitor 2A). In vitro Sox9 showed mechanosensitive responses with increased expression and nuclear translocation in senescent cells and on stiff matrices. Sox9 was found to regulate ECM stiffness and organization by orchestrating changes in collagen (Col) expression and reducing VSMC contractility, leading to the formation of an ECM that mirrored that of senescent cells. These ECM changes promoted phenotypic modulation of VSMCs, whereby senescent cells plated on ECM synthesized from cells depleted of Sox9 returned to a proliferative state, while proliferating cells on a matrix produced by Sox9 expressing cells showed reduced proliferation and increased DNA damage, reiterating features of senescent cells. LH3 (procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3) was identified as an Sox9 target and key regulator of ECM stiffness. LH3 is packaged into extracellular vesicles and Sox9 promotes extracellular vesicle secretion, leading to increased LH3 deposition within the ECM. CONCLUSIONS: These findings highlight the crucial role of ECM structure and composition in regulating VSMC phenotype. We identify a positive feedback cycle, whereby cellular senescence and increased ECM stiffening promote Sox9 expression, which, in turn, drives further ECM modifications to further accelerate stiffening and senescence.

Emerging role of lipophagy in liver disorders.

Lipophagy is a selective degradation of lipids by a lysosomal-mediated pathway, and dysregulation of lipophagy is linked with the pathological hallmark of many liver diseases. Downregulation of lipophagy in liver cells results in abnormal accumulation of LDs (Lipid droplets) in hepatocytes which is a characteristic feature of several liver pathologies such as nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Contrarily, upregulation of lipophagy in activated hepatic stellate cells (HSCs) is associated with hepatic fibrosis and cirrhosis. Lipid metabolism reprogramming in violent cancer cells contributes to the progression of liver cancer. In this review, we have summarized the recent studies focusing on various components of the lipophagic machinery that can be modulated for their potential role as therapeutic agents against a wide range of liver diseases.

Recent Progress in Azobenzene-Based Supramolecular Materials and Applications.

Azobenzene-containing small molecules and polymers are functional photoswitchable molecules to form supramolecular nanomaterials for various applications. Recently, supramolecular nanomaterials have received enormous attention in material science because of their simple bottom-up synthesis approach, understandable mechanisms and structural features, and batch-to-batch reproducibility. Azobenzene is a light-responsive functional moiety in the molecular design of small molecules and polymers and is used to switch the photophysical properties of supramolecular nanomaterials. Herein, we review the latest literature on supramolecular nano- and micro-materials formed from azobenzene-containing small molecules and polymers through the combinatorial effect of weak molecular interactions. Different classes including complex coacervates, host-guest systems, co-assembled, and self-assembled supramolecular materials, where azobenzene is an essential moiety in small molecules, and photophysical properties are discussed. Afterward, azobenzene-containing polymers-based supramolecular photoresponsive materials formed through the host-guest approach, polymerization-induced self-assembly, and post-polymerization assembly techniques are highlighted. In addition to this, the applications of photoswitchable supramolecular materials in pH sensing, and CO2 capture are presented. In the end, the conclusion and future perspective of azobenzene-based supramolecular materials for molecular assembly design, and applications are given.

Full Study Report of Andexanet Alfa for Bleeding Associated with Factor Xa Inhibitors.

BACKGROUND: Andexanet alfa is a modified recombinant inactive form of human factor Xa developed for reversal of factor Xa inhibitors. METHODS: We evaluated 352 patients who had acute major bleeding within 18 hours after administration of a factor Xa inhibitor. The patients received a bolus of andexanet, followed by a 2-hour infusion. The coprimary outcomes were the percent change in anti-factor Xa activity after andexanet treatment and the percentage of patients with excellent or good hemostatic efficacy at 12 hours after the end of the infusion, with hemostatic efficacy adjudicated on the basis of prespecified criteria. Efficacy was assessed in the subgroup of patients with confirmed major bleeding and baseline anti-factor Xa activity of at least 75 ng per milliliter (or ≥0.25 IU per milliliter for those receiving enoxaparin). RESULTS: Patients had a mean age of 77 years, and most had substantial cardiovascular disease. Bleeding was predominantly intracranial (in 227 patients [64%]) or gastrointestinal (in 90 patients [26%]). In patients who had received apixaban, the median anti-factor Xa activity decreased from 149.7 ng per milliliter at baseline to 11.1 ng per milliliter after the andexanet bolus (92% reduction; 95% confidence interval [CI], 91 to 93); in patients who had received rivaroxaban, the median value decreased from 211.8 ng per milliliter to 14.2 ng per milliliter (92% reduction; 95% CI, 88 to 94). Excellent or good hemostasis occurred in 204 of 249 patients (82%) who could be evaluated. Within 30 days, death occurred in 49 patients (14%) and a thrombotic event in 34 (10%). Reduction in anti-factor Xa activity was not predictive of hemostatic efficacy overall but was modestly predictive in patients with intracranial hemorrhage. CONCLUSIONS: In patients with acute major bleeding associated with the use of a factor Xa inhibitor, treatment with andexanet markedly reduced anti-factor Xa activity, and 82% of patients had excellent or good hemostatic efficacy at 12 hours, as adjudicated according to prespecified criteria. (Funded by Portola Pharmaceuticals; ANNEXA-4 ClinicalTrials.gov number, NCT02329327.).

Runx2 (Runt-Related Transcription Factor 2) Links the DNA Damage Response to Osteogenic Reprogramming and Apoptosis of Vascular Smooth Muscle Cells.

[Figure: see text].

Endoplasmic Reticulum Stress Mediates Vascular Smooth Muscle Cell Calcification via Increased Release of Grp78 (Glucose-Regulated Protein, 78 kDa)-Loaded Extracellular Vesicles.

OBJECTIVE: Vascular calcification is common among aging populations and mediated by vascular smooth muscle cells (VSMCs). The endoplasmic reticulum (ER) is involved in protein folding and ER stress has been implicated in bone mineralization. The role of ER stress in VSMC-mediated calcification is less clear. Approach and Results: mRNA expression of the ER stress markers PERK (PKR (protein kinase RNA)-like ER kinase), ATF (activating transcription factor) 4, ATF6, and Grp78 (glucose-regulated protein, 78 kDa) was detectable in human vessels with levels of PERK decreased in calcified plaques compared to healthy vessels. Protein deposition of Grp78/Grp94 was increased in the matrix of calcified arteries. Induction of ER stress accelerated human primary VSMC-mediated calcification, elevated expression of some osteogenic markers (Runx2 [RUNX family transcription factor 2], OSX [Osterix], ALP [alkaline phosphatse], BSP [bone sialoprotein], and OPG [osteoprotegerin]), and decreased expression of SMC markers. ER stress potentiated extracellular vesicle (EV) release via SMPD3 (sphingomyelin phosphodiesterase 3). EVs from ER stress-treated VSMCs showed increased Grp78 levels and calcification. Electron microscopy confirmed the presence of Grp78/Grp94 in EVs. siRNA (short interfering RNA) knock-down of Grp78 decreased calcification. Warfarin-induced Grp78 and ATF4 expression in rat aortas and VSMCs and increased calcification in an ER stress-dependent manner via increased EV release. CONCLUSIONS: ER stress induces vascular calcification by increasing release of Grp78-loaded EVs. Our results reveal a novel mechanism of action of warfarin, involving increased EV release via the PERK-ATF4 pathway, contributing to calcification. This study is the first to show that warfarin induces ER stress and to link ER stress to cargo loading of EVs.

Expression levels of MiRNA-16, SURVIVIN and TP53 in Preeclamptic and Normotensive women.

OBJECTIVE: To measure and compare micro ribonucleic acid-16, survivin and tumour protein p53-regulated apoptosis-inducing protein 1 expression levels in preeclamptic and normotensive pregnancies, and to check the correlation of micro ribonucleic acid-16 with messenger ribonucleic acid expression of survivin and tumour protein p53. METHODS: The observational cross-sectional comparative study was conducted at the Department of Physiology and Cell Biology, University of Health Sciences, Lahore, Pakistan, from 2016 to 2018, and comprised preeclamptic women in group A and normotensive women in group B. The preeclamptic patients were further categorised into early-onset preeclampsia subgroup A1and late-onset preeclampsia group A2. Expression of micro ribonucleic acid-16, messenger ribonucleic acid expression of survivin and tumour protein p53 in preeclamptic and normotensive pregnancies were analysed using real time polymerase chain reaction. Data was analysed using SPSS 22. RESULTS: Of the 54 patients, 27(50%) were in each of the two groups. Within group A, 14(52%) patients were in group A1 and 13(48%) in group A2. The expression of micro ribonucleic acid 16 showed significant increase in group A compared to group B (p<0.05). The difference was not significant between the subgroups A1 and A2. The levels of messenger ribonucleic acid expression of survivin and tumour protein p53 were deregulated in group A, with a decrease in survivin and an increase in tumour protein p53. The messenger ribonucleic acid expression of survivin and tumour protein p53 showed statistically significant differences across subgroups A1 and A2 (p<0.05). The micro ribonucleic acid-16 expression correlated negatively with messenger ribonucleic acid expression of survivin, but exhibited a positive correlation with tumour protein p53. CONCLUSIONS: Deregulated micro ribonucleic acid-16 along with differentially expressed apoptotic genes, survivin and tumour protein p53 might result in altered apoptosis implicated in the pathogenesis of preeclampsia.

Arterial "inflammaging" drives vascular calcification in children on dialysis.

Children on dialysis have a cardiovascular mortality risk equivalent to older adults in the general population, and rapidly develop medial vascular calcification, an age-associated pathology. We hypothesized that premature vascular ageing contributes to calcification in children with advanced chronic kidney disease (CKD). Vessels from children with Stage 5 CKD with and without dialysis had evidence of increased oxidative DNA damage. The senescence markers p16 and p21 were also increased in vessels from children on dialysis. Treatment of vessel rings ex vivo with calcifying media increased oxidative DNA damage in vessels from children with Stage 5 CKD, but not in those from healthy controls. Vascular smooth muscle cells cultured from children on dialysis exhibited persistent DNA damage, impaired DNA damage repair, and accelerated senescence. Under calcifying conditions vascular smooth muscle cells from children on dialysis showed increased osteogenic differentiation and calcification. These changes correlated with activation of the senescence-associated secretory phenotype (SASP), an inflammatory phenotype characterized by the secretion of proinflammatory cytokines and growth factors. Blockade of ataxia-telangiectasia mutated (ATM)-mediated DNA damage signaling reduced both inflammation and calcification. Clinically, children on dialysis had elevated circulating levels of osteogenic SASP factors that correlated with increased vascular stiffness and coronary artery calcification. These data imply that dysregulated mineral metabolism drives vascular "inflammaging" by promoting oxidative DNA damage, premature senescence, and activation of a pro-inflammatory SASP. Drugs that target DNA damage signaling or eliminate senescent cells may have the potential to prevent vascular calcification in patients with advanced CKD.

Association of MIR146A rs2910164 variation with a predisposition to sporadic breast cancer in a Pakistani cohort.

Single-nucleotide polymorphisms (SNPs) in genes coding for microRNAs (miRNAs) play a pivotal role in the progression of breast cancer (BC). We investigated the association of miR-146a rs2910164 GC polymorphism with the risk of BC in the Pakistani population. The miR-146a rs2910164 polymorphism was genotyped in 300 BC cases and 300 age- and gender-matched healthy controls using T-ARMS-PCR. Genotype and allele frequencies were calculated and the association between genotypes and the risk of BC was calculated by odds ratio (OR) and confidence interval (95%). A significant difference in genotypic frequencies (χ2  = 63.10; P = <0.0001) and allelic frequencies (OR = 0.3955 (0.3132-0.4993); P = < 0.0001) was observed between cases and controls. Furthermore, we also found that miR-146 rs2910164 CC homozygote increased the risk of BC in the dominant (OR = 0.2397 (0.1629-0.3526); P = 0.0001; GG vs. GC + CC) and recessive (OR = 2.803 (1.865-4.213); P = <0.0001; CC vs. GC + GG) inheritance models. In summary, miR-146a rs2910164 GC is significantly associated with BC in the Pakistani population. To our knowledge, this is the first study that assessed MIR146a rs2910164 G > C SNP in Pakistani population. By analyzing the secondary structure of MIR146A variant, a significant structural modification was noted. Study with a larger sample size is needed to further confirm of these findings.

Host attachment and fluid shear are integrated into a mechanical signal regulating virulence in Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen causing hemorrhagic colitis and hemolytic uremic syndrome. EHEC colonizes the intestinal tract through a range of virulence factors encoded by the locus of enterocyte effacement (LEE), as well as Shiga toxin. Although the factors involved in colonization and disease are well characterized, how EHEC regulates its expression in response to a host encounter is not well understood. Here, we report that EHEC perceives attachment to host cells as a mechanical cue that leads to expression of LEE-encoded virulence genes. This signal is transduced via the LEE-encoded global regulator of LEE-encoded regulator (Ler) and global regulator of Ler and is further enhanced by levels of shear force similar to peristaltic forces in the intestinal tract. Our data suggest that, in addition to a range of chemical environmental signals, EHEC is capable of sensing and responding to mechanical cues to adapt to its host's physiology.

Sofosbuvir For The Treatment Of Hepatitis C Genotype 3 Infected Patients In Pakistan.

BACKGROUND: This study was conducted to determine the viral responses of patients with chronic infection of Hepatitis C virus treated with sofubuvir. METHODS: This Quasi experimental study was conducted at Centre for Liver and Digestive Diseases, Holy Family Hospital, Rawalpindi from September 2014 to September 2016. 502 patients with HCV genotype 3 including treatment naive, non-responders or relapsers to previous interferon based therapy along with patients having decompensated cirrhosis (child class B or C) were included in the study. All patients were treated with Sofosbuvir 400 mg once daily along with Ribavirin for 6 months. Follow-up qualitative PCR (polymerase Chain Reaction) were performed at 4 weeks interval to assess RVR (Rapid virological Response), end of treatment to determine ETR (End of treatment response) and 3 months post treatment to determine SVR12 (Sustained viral response at 12 week). RESULTS: 91% of the patients had become PCR negative at completion of four weeks of treatment with Sofosbuvir, whereas at completion of treatment 96.5% had attained a negative PCR. Sustained virological response at 12 weeks post therapy (SVR12) was attained in 85.5% of patients. No statistically significant associations were found with attainment status of RVR, ETR and SVR based on previous treatment status or presence of Decompensated liver disease. However, attainment of SVR was slightly more in females (p value=0.03). The serological profiles of patients whether they attained PCR at week 4, 24 of treatment or 12 weeks' post treatment did not exhibit any statistically significant difference. CONCLUSION: Sofosbuvir is effective in eradicating hepatitis C virus irrespective of previous treatment or liver fibrosis status in genotype 3 HCV Pakistani patients.

Facile synthesis of 1-alkoxy-1H-benzo- and 7-azabenzotriazoles from peptide coupling agents, mechanistic studies, and synthetic applications.

(1H-Benzo[d][1,2,3]triazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), 1H-benzo[d][1,2,3]triazol-1-yl 4-methylbenzenesulfonate (Bt-OTs), and 3H-[1,2,3]triazolo[4,5-b]pyridine-3-yl 4-methylbenzenesulfonate (At-OTs) are classically utilized in peptide synthesis for amide-bond formation. However, a previously undescribed reaction of these compounds with alcohols in the presence of a base, leads to 1-alkoxy-1H-benzo- (Bt-OR) and 7-azabenzotriazoles (At-OR). Although BOP undergoes reactions with alcohols to furnish 1-alkoxy-1H-benzotriazoles, Bt-OTs proved to be superior. Both, primary and secondary alcohols undergo reaction under generally mild reaction conditions. Correspondingly, 1-alkoxy-1H-7-azabenzotriazoles were synthesized from At-OTs. Mechanistically, there are three pathways by which these peptide-coupling agents can react with alcohols. From (31)P{(1)H}, [(18)O]-labeling, and other chemical experiments, phosphonium and tosylate derivatives of alcohols seem to be intermediates. These then react with BtO(-) and AtO(-) produced in situ. In order to demonstrate broader utility, this novel reaction has been used to prepare a series of acyclic nucleoside-like compounds. Because BtO(-) is a nucleofuge, several Bt-OCH2Ar substrates have been evaluated in nucleophilic substitution reactions. Finally, the possible formation of Pd π-allyl complexes by departure of BtO(-) has been queried. Thus, alpha-allylation of three cyclic ketones was evaluated with 1-(cinnamyloxy)-1H-benzo[d][1,2,3]triazole, via in situ formation of pyrrolidine enamines and Pd catalysis.

Surface crafting and entrapment of CsPbBr3 perovskite QDs in ZIF-8 for ammonia recognition.

The substantial optical features of perovskite quantum dots (PQD) lead to rapid growth in the investigation of their surface and lattice doping for optoelectronic and biochemical sensor advancements. Herein, we have used the surface ligand crafting model of PQD by ammonia and its optimum response to recognise ammonia in the sensing cellulose paper. The PQD with acetyl amine and octanoic acid capped were synthesized and entrapped in zeolites imidazole framework to delay the instant quenching and envisaged response to ammonia with high sensitivity. The hybrid perovskite quantum dots and Zeolite imidazolate framework-8 (PQD@ZIF-8) materials were further immersed in cellulose paper for solid-state sensor fabrication for the detection of ammonia by naked-eye and a Xiaomi Note-5 mobile camera. The ammonia was measured with high sensitivity at ambient conditions, with a detection limit of 16 ppm and a linear detection range of 1 to 500 ppm. This research provides a new platform for designing sensor selectivity and sensitivity, which could be used to further develop fluorescent nanomaterials-based sensors for small molecule detection.

Vascular smooth muscle cell senescence accelerates medin aggregation via small extracellular vesicle secretion and extracellular matrix reorganization.

Vascular amyloidosis, caused when peptide monomers aggregate into insoluble amyloid, is a prevalent age-associated pathology. Aortic medial amyloid (AMA) is the most common human amyloid and is composed of medin, a 50-amino acid peptide. Emerging evidence has implicated extracellular vesicles (EVs) as mediators of pathological amyloid accumulation in the extracellular matrix (ECM). To determine the mechanisms of AMA formation with age, we explored the impact of vascular smooth muscle cell (VSMC) senescence, EV secretion, and ECM remodeling on medin accumulation. Medin was detected in EVs secreted from primary VSMCs. Small, round medin aggregates colocalized with EV markers in decellularized ECM in vitro and medin was shown on the surface of EVs deposited in the ECM. Decreasing EV secretion with an inhibitor attenuated aggregation and deposition of medin in the ECM. Medin accumulation in the aortic wall of human subjects was strongly correlated with age and VSMC senescence increased EV secretion, increased EV medin loading and triggered deposition of fibril-like medin. Proteomic analysis showed VSMC senescence induced changes in EV cargo and ECM composition, which led to enhanced EV-ECM binding and accelerated medin aggregation. Abundance of the proteoglycan, HSPG2, was increased in the senescent ECM and colocalized with EVs and medin. Isolated EVs selectively bound to HSPG2 in the ECM and its knock-down decreased formation of fibril-like medin structures. These data identify VSMC-derived EVs and HSPG2 in the ECM as key mediators of medin accumulation, contributing to age-associated AMA development.

Prelamin A mediates myocardial inflammation in dilated and HIV-associated cardiomyopathies.

Cardiomyopathies are complex heart muscle diseases that can be inherited or acquired. Dilated cardiomyopathy can result from mutations in LMNA, encoding the nuclear intermediate filament proteins lamin A/C. Some LMNA mutations lead to accumulation of the lamin A precursor, prelamin A, which is disease causing in a number of tissues, yet its impact upon the heart is unknown. Here, we discovered myocardial prelamin A accumulation occurred in a case of dilated cardiomyopathy, and we show that a potentially novel mouse model of cardiac-specific prelamin A accumulation exhibited a phenotype consistent with inflammatory cardiomyopathy, which we observed to be similar to HIV-associated cardiomyopathy, an acquired disease state. Numerous HIV protease therapies are known to inhibit ZMPSTE24, the enzyme responsible for prelamin A processing, and we confirmed that accumulation of prelamin A occurred in HIV+ patient cardiac biopsies. These findings (a) confirm a unifying pathological role for prelamin A common to genetic and acquired cardiomyopathies; (b) have implications for the management of HIV patients with cardiac disease, suggesting protease inhibitors should be replaced with alternative therapies (i.e., nonnucleoside reverse transcriptase inhibitors); and (c) suggest that targeting inflammation may be a useful treatment strategy for certain forms of inherited cardiomyopathy.

Poly(ADP-Ribose) Links the DNA Damage Response and Biomineralization.

Biomineralization of the extracellular matrix is an essential, regulated process. Inappropriate mineralization of bone and the vasculature has devastating effects on patient health, yet an integrated understanding of the chemical and cell biological processes that lead to mineral nucleation remains elusive. Here, we report that biomineralization of bone and the vasculature is associated with extracellular poly(ADP-ribose) synthesized by poly(ADP-ribose) polymerases in response to oxidative and/or DNA damage. We use ultrastructural methods to show poly(ADP-ribose) can form both calcified spherical particles, reminiscent of those found in vascular calcification, and biomimetically calcified collagen fibrils similar to bone. Importantly, inhibition of poly(ADP-ribose) biosynthesis in vitro and in vivo inhibits biomineralization, suggesting a therapeutic route for the treatment of vascular calcifications. We conclude that poly(ADP-ribose) plays a central chemical role in both pathological and physiological extracellular matrix calcification.

Spectrum of primary non-cutaneous malignant melanomas in northern Pakistan.

OBJECTIVE: To study the frequency and distribution of non-cutaneous malignant melanoma in Northern Pakistan using a cross-sectional descriptive design at the Armed Forces Institute of Pathology Rawalpindi, from 1987-2008. MATERIALS AND METHODS: A total of 234 cases of malignant melanoma reported from 1987-2008 were retrieved from the AFIP tumour registry and analysed for age, gender and site using computer software SPSS 12. RESULTS: Of a total of 58,680 malignant cases reported at AFIP from 1987-2008, only 234 (0.4%) were of malignant melanoma, 142 in males and 92 in females. The age range was 10-97 years, with a mean of 53 ± 16. Only 69 (29.5%) were cutaneous and 165 (70.5%) were non-cutaneous in origin: there were 62 (37.4%) cases from anorectal region, 39 (23.4%)were from eye ball and 17 (10.2%) in the nasopharynx, 13 cases (7.8%) from oral mucosa and 11 (6.6%) from the vagina. CONCLUSION: Malignant melanoma is markedly less cutaneous in Pakistan as compared to the western world, often being found in the anorectal region followed by the eye, nasopharynx, and oral mucosa in descending order.

Standardization of pathology laboratories in Pakistan: problems and prospects.

A Total Quality Management System with an internationally recognized accreditation process is the only guarantee of a reliable pathology service. However in a developing country like Pakistan nearly 90% of labs are small and without adequate physical and man power infrastructures. A modified plan may have to be tailored for them. A two tier system has been formulated. Accreditation based on ISO 15189 is to be introduced on voluntary basis. The labs which do not qualify for international standardization would be brought into quality net through a registration process. All such labs will be initiated into a simplified quality management system. Participation in proficiency testing program will be mandatory. They will be provided education and training to become eligible for accreditation. It is hoped that this simplified system will provide an impetus for evolving a workable and comprehensive mechanism through which pathology laboratories in the country will be able to offer better and more reliable services.