FOXC1 regulates endothelial CD98 (LAT1/4F2hc) expression in retinal angiogenesis and blood-retina barrier formation.

Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is essential for the development of new organ systems, but transcriptional control of angiogenesis remains incompletely understood. Here we show that FOXC1 is essential for retinal angiogenesis. Endothelial cell (EC)-specific loss of Foxc1 impairs retinal vascular growth and expression of Slc3a2 and Slc7a5, which encode the heterodimeric CD98 (LAT1/4F2hc) amino acid transporter and regulate the intracellular transport of essential amino acids and activation of the mammalian target of rapamycin (mTOR). EC-Foxc1 deficiency diminishes mTOR activity, while administration of the mTOR agonist MHY-1485 rescues perturbed retinal angiogenesis. EC-Foxc1 expression is required for retinal revascularization and resolution of neovascular tufts in a model of oxygen-induced retinopathy. Foxc1 is also indispensable for pericytes, a critical component of the blood-retina barrier during retinal angiogenesis. Our findings establish FOXC1 as a crucial regulator of retinal vessels and identify therapeutic targets for treating retinal vascular disease.

Identification and characterization of a novel variant in C-terminal region of Antithrombin (Ala427Thr) associated with type II AT deficiency leading to polymer formation.

Antithrombin (AT) deficiency is a rare but strong risk factor for the thrombosis development. Mutations in gene encoding AT (SERPINC1) have provided a detailed understanding of AT deficiency and subsequent development of thrombotic complications. In the present study, we describe a case of thrombotic patient with reduced AT activity and normal AT antigen levels. AT deficiency in the patient was explained by the presence of heterozygous mutation g.13397A>G (Ala427Thr) in exon 6 of SERPINC1. Reduced APTT and TT with normal PT were observed. The mutation was found to be absent in healthy controls (n = 62). In vitro purification and characterization of variant AT showed significant decrease in fluorescence emission intensity, decreased bis-ANS fluorescence emission, changes in secondary structure and presence of polymerized AT in patient's plasma as assessed by fluorescence, circular dichroism and transmission electron microscopy respectively. Furthermore, molecular dynamics simulation studies showed altered conformation due to Ala427Thr substitution. Our study shows that genetic screening should be carried out in AT deficient patients in addition to the routinely used functional assays to understand the molecular basis of disease development.

Elevated surface-bound complement FH alters the function of platelets and monocytes in FHR1/3 null healthy individuals.

Complement factor H (FH) and FH-related proteins (FHRs), structurally similar proteins are involved in the regulation of complement activation. Homozygous deletion of FHR 1 and 3 proteins (FHR1/3-/-) is known as a risk factor for disorders such as aHUS and SLE, characterised by thrombo-inflammatory complications. Interestingly, FHR1/3-/- genotype also exists as polymorphism in healthy population of various ethnicities around the world including 8-10% Indians. In an effort to understand the functional role of this polymorphism, we describe in this study an elevated surface-bound FH on platelets and monocytes, but not other blood cells in FHR1/3 -/- healthy individuals. The FHR1/3-/- platelets displayed diminish ability to form aggregates in response to agonists in vitro. The FHR1/3-/- monocytes displayed elevated secretion of TNFα, IL1ß, IL6 and IL10 in response to TLR ligands. However, exogenous FH limits platelet aggregates formation as well as cytokine secretion in monocytes. Therefore, observations together suggest a differential regulation of platelets and monocytes by FH-FHR1/3 axis in healthy individuals. While these findings will need more detailed investigation, it is clear that the connection between FH-FHR axis and thrombo-inflammatory complications is likely to be complex in diseases including aHUS and SLE, and provide interesting new directions for future study.

Unique case of autoantibody mediated inactivation of ADAMTS13 in an Indian TTP patient.

A young Indian female visited hospital as a suspected case of thrombotic thrombocytopenic purpura (TTP) with relapsed thrombotic complications with low platelet counts, infarct in middle cerebral artery and thrombi in microvessels. We first confirmed the deficiency of ADAMTS13 metalloprotease in this patient showing improper cleavage of vWF multimers by her plasma unlike her parents and brother. Although patient had very less ADAMTS13 antigen in plasma, but it did not appear to be the cause of deficiency of the enzyme, because her father had similarly low antigen level and he never had prothrombotic complications. While investigating the genetic change in ADAMTS13, we observed four homozygous-SNPs (g.420T>C, g.1342C>G, g.1716G>A and g.2280T>C) in exon 5, 12, 15 and 19 respectively in patient and her father unlike the heterozygous form of same SNPs in mother and brother. Further to investigate the cause of ADAMTS13 deficiency, we observed an elevated level of antibody against ADAMTS13 in patient unlike her father and other family members. Our study therefore provides the molecular approach of diagnosis of TTP in this patient and also highlights the use of such techniques in India. More importantly, study provides the clue of alternate treatment such as immunosuppressant therapy to this patient.

Absence of Nonclassical Monocytes in Hemolytic Patients: Free Hb and NO-Mediated Mechanism.

In a recent work, we have described the kinetics among the monocyte subsets in the peripheral blood of hemolytic patients including paroxysmal nocturnal hemoglobinuria (PNH) and sickle cell disease (SCD). After engulfing Hb-activated platelets, classical monocytes (CD14+CD16-) significantly transformed into highly inflammatory (CD14+CD16hi) subsets in vitro. An estimated 40% of total circulating monocytes in PNH and 70% in SCD patients existed as CD14+CD16hi subsets. In this study, we show that the nonclassical (CD14dimCD16+) monocyte subsets are nearly absent in patients with PNH or SCD, compared to 10-12% cells in healthy individuals. In mechanism, we have described the unique role of both free Hb and nitric oxide (NO) in reducing number of nonclassical subsets more than classical monocytes. After engulfing Hb-activated platelets, the monocytes including nonclassical subsets acquired rapid cell death within 12 h in vitro. Further, the treatment to monocytes either with the secretome of Hb-activated platelets containing NO and free Hb or purified free Hb along with GSNO (a physiological NO donor) enhanced rapid cell death. Besides, our data from both PNH and SCD patients exhibited a direct correlation between intracellular NO and cell death marker 7AAD in monocytes from the peripheral blood. Our data together suggest that due to the immune surveillance nature, the nonclassical or patrolling monocytes are encountered frequently by Hb-activated platelets, free Hb, and NO in the circulation of hemolytic patients and are predisposed to die rapidly.

Platelet factor 4 promotes rapid replication and propagation of Dengue and Japanese encephalitis viruses.

BACKGROUND: Activated platelets release cytokines/proteins including CXCL4 (PF4), CCL5 and fibrinopeptides, which regulate infection of several pathogenic viruses such as HIV, H1N1 and HCV in human. Since platelet activation is the hallmark of Dengue virus (DV) infection, we investigated the role of platelets in DV replication and also in a closely related Japanese Encephalitis virus (JEV). METHODS AND FINDINGS: Microscopy and PCR analysis revealed a 4-fold increase in DV replication in primary monocytes or monocytic THP-1 cells in vitro upon incubation with either DV-activated platelets or supernatant from DV-activated platelets. The mass spectrometry based proteomic data from extra-nuclear fraction of above THP-1 lysate showed the crucial association of PF4 with enhanced DV replication. Our cytokine analysis and immunoblot assay showed significant inhibition of IFN-α production in monocytes via p38MAPK-STAT2-IRF9 axis. Blocking PF4 through antibodies or its receptor CXCR3 through inhibitor i.e. AMG487, significantly rescued production of IFN-α resulting in potent inhibition of DV replication in monocytes. Further, flow cytometry and ELISA data showed the direct correlation between elevated plasma PF4 with increased viral NS1 in circulating monocytes in febrile DV patients at day-3 of fever than day-9. Similarly, PF4 also showed direct effects in promoting the JEV replication in monocytes and microglia cells in vitro. The in vitro results were also validated in mice, where AMG487 treatment significantly improved the survival of JEV infected animals. INTERPRETATION: Our study suggests that PF4-CXCR3-IFN axis is a potential target for developing treatment regimen against viral infections including JEV and DV.

Identification of 2 Novel Polymorphisms and rs3138521 in 5' Untranslated Region of SERPINC1 Gene in North Indian Population With Deep Vein Thrombosis.

Antithrombin III (AT) is the most important endogenous anticoagulant, and genetic variability in SERPINC1, gene encoding AT, is low. Mutations leading to AT deficiency and increased thrombotic risk are well known; however, only 2 studies have reported mutations in regulatory region of SERPINC1 gene till date. Aim of the present study was to identify genetic variations in SERPINC1 5' untranslated region (UTR) in Indian patients with deep vein thrombosis (DVT) having AT deficiency. DNA sequencing was used to identify underlying genetic defects in SERPINC1 regulatory region. In silico tools TFBIND and PROMO were used to identify transcription factor binding sites in the promoter region. We have identified 2 novel polymorphisms, g.25G>A and g.-1A>T, and 2 known g.67G>A and rs3138521 5' UTR polymorphisms in SERPINC1 regulatory region in Indian patients with DVT for the first time. In present study, allele frequencies of rs3138521 (S: 0.37 and F: 0.63) were similar to that reported in Western population and were not associated with low plasma AT levels ( P value .5). This is the first report of regulatory region polymorphisms in SERPINC1 gene in Indian population. Our results strongly suggest that similar studies should be included when ever no mutation is detected in protein-coding region of AT gene.

Prevalence of Factor V Genetic Variants Associated With Indian APCR Contributing to Thrombotic Risk.

Phenotypic resistance to activated protein C (APC) is a complex mechanism associated with increased thrombosis risk. Activated protein C resistance (APCR) is mainly influenced by FVLeiden mutation, and various other single nucleotide polymorphisms (SNPs) in FV gene are known to be associated with APCR. The aim of present study was to investigate the incidence and assess possible mechanisms of APCR in Indian patients with deep vein thrombosis (DVT). Three hundred and ten Doppler-proven patients with DVT were screened for APCR, and 50 APCR positive patients and 50 controls were typed for FVLeiden, Hong Kong, Cambridge, HR2 haplotype, Glu666Asp, Ala485Lys, and Liverpool using either polymerase chain reaction (PCR)-restriction fragment length polymorphism or allele specific PCR. FVLeiden was commonest cause of APCR (50%) in Indian patients with DVT being statistically significant ( P = .001) compared to controls. FV Liverpool, FV Glu666Asp and FV Ala485Lys were studied for the first time in Indian population. FV Liverpool, FV Glu666Asp, Hong Kong, and Cambridge were found to be absent. High frequency of Ala485Lys in patients shows that it might be a risk factor contributing to APCR in Indian patients with DVT. HR2 haplotype was not associated with APCR; however, presence of homozygous HR2 haplotype in patients only indicates the role it might play in Indian APCR population. In conclusion, contribution of FVLeiden causing APCR in Indian population is not as strong as previously reported in Western countries. The presence of other SNPs observed in the present study requires such studies on larger sample size to understand the molecular basis of defect.

Role of heparin and non heparin binding serpins in coagulation and angiogenesis: A complex interplay.

Pro-coagulant, anti-coagulant and fibrinolytic pathways are responsible for maintaining hemostatic balance under physiological conditions. Any deviation from these pathways would result in hypercoagulability leading to life threatening diseases like myocardial infarction, stroke, portal vein thrombosis, deep vein thrombosis (DVT) and pulmonary embolism (PE). Angiogenesis is the process of sprouting of new blood vessels from pre-existing ones and plays a critical role in vascular repair, diabetic retinopathy, chronic inflammation and cancer progression. Serpins; a superfamily of protease inhibitors, play a key role in regulating both angiogenesis and coagulation. They are characterized by the presence of highly conserved secondary structure comprising of 3 ß-sheets and 7-9 α-helices. Inhibitory role of serpins is modulated by binding to cofactors, specially heparin and heparan sulfate proteoglycans (HSPGs) present on cell surfaces and extracellular matrix. Heparin and HSPGs are the mainstay of anti-coagulant therapy and also have therapeutic potential as anti-angiogenic inhibitors. Many of the heparin binding serpins that regulate coagulation cascade are also potent inhibitors of angiogenesis. Understanding the molecular mechanism of the switch between their specific anti-coagulant and anti-angiogenic role during inflammation, stress and regular hemostasis is important. In this review, we have tried to integrate the role of different serpins, their interaction with cofactors and their interplay in regulating coagulation and angiogenesis.

Antithrombin III deficiency in Indian patients with deep vein thrombosis: identification of first India based AT variants including a novel point mutation (T280A) that leads to aggregation.

Antithrombin III (AT) is the main inhibitor of blood coagulation proteases like thrombin and factor Xa. In this study we report the identification and characterization of several variants of AT for the first time in Indian population. We screened 1950 deep vein thrombosis (DVT) patients for AT activity and antigen levels. DNA sequencing was further carried out in patients with low AT activity and/or antigen levels to identify variations in the AT gene. Two families, one with type I and the other with type II AT deficiency were identified. Three members of family I showed an increase in the coagulation rates and recurrent thrombosis in this family was solely attributed to the rs2227589 polymorphism. Four members of family II spanning two generations had normal antigen levels and decreased AT activity. A novel single nucleotide insertion, g.13362_13363insA in this family in addition to g.2603T>C (p.R47C) mutation were identified. AT purified from patient's plasma on hi-trap heparin column showed a marked decrease in heparin affinity and thrombin inhibition rates. Western blot analysis showed the presence of aggregated AT. We also report a novel point mutation at position g.7549 A>G (p.T280A), that is highly conserved in serpin family. Variant protein isolated from patient plasma indicated loss of regulatory function due to in-vivo polymerization. In conclusion this is the first report of AT mutations in SERPINC1 gene in Indo-Aryan population where a novel point mutation p.T280A and a novel single nucleotide insertion g.13362_13363insA are reported in addition to known variants like p.R47C, p.C4-X and polymorphisms of rs2227598, PstI and DdeI.

Polymorphisms in factor V and antithrombin III gene in recurrent pregnancy loss: a case-control study in Indian population.

Recurrent pregnancy loss (RPL) can be caused due to diverse factors with thrombophilia being one of them. The association of various thrombophilic risk factors with RPL is inconsistent in different studies and the frequency of these risk factors in Indian population is obscure. Five hundred and eighty patients with either recurrent early miscarriage or a history of at least one late miscarriage were screened for deficiency of protein C (PC), protein S (PS), antithrombin III (AT), APC resistance and prothrombin 20210G > A mutation. APC resistance positive patients were typed for the factor V Leiden, factor V Hong Kong/Cambridge mutations, and HR2 haplotype. PstI and rs2227589 AT mutations were detected by direct sequencing. APC resistance (13.4 %) was detected to be most common in Indian RPL patients followed by PS (10.6 %), PC (9.8 %) and AT deficiency (4.31 %.). FV Leiden was shown to be associated with APC resistance while HR2 haplotype was not associated with APC resistance (p values: 0.0001 and 0.327 respectively) and the increased risk of RPL. PstI and rs2227589 polymorphisms were similar in patients and controls and not associated with AT deficiency in RPL. Our study emphasizes the presence of other contributory factors towards APC resistance rather than FV Leiden alone. This is the first Indian study where HR2 haplotype and rs2227589 are observed to be present in RPL population. Although not significant, occurrence of rs2227589 and FV HR2 in homozygous condition necessitates the study of these polymorphisms in a larger sample size.