Roby J. Jose

Diagnosis of herpes simplex virus-1 keratitis: comparison of Giemsa stain, immunofluorescence assay and polymerase chain reaction.

Purpose. To evaluate three different diagnostic tests against the gold standard of viral isolation, in the diagnosis of HSV-1 keratitis. Methods. Corneal scrapings from 170 patients with clinically suspected HSV keratitis were tested by; 1) Giemsa staining procedure for the presence of multinucleated giant cells and lymphocytes, 2) immunofluorescence assay for HSV-1 antigen, 3) polymerase chain reaction (PCR) for HSV-1 DNA and 4) virus isolation by shell vial culture in SIRC (Rabbit corneal epithelial cell line). The results of the former three tests were compared among 14 cases that were culture positive and 156 cases that were culture negative for HSV-1. Results. The sensitivity of PCR was 100%, while IFA and Giemsa had sensitivities of 85.7% and 57.1% respectively. The specificity of PCR, IFA and Giemsa were found to be 67.9%, 85.3% and 85.9% respectively. Conclusions. In the present study, a combination of PCR and immunofluorescence assay appears to be the most suitable choice of tests for diagnosis of HSV-1 keratitis, while detection of MNGC by Giemsa staining procedure may give us a presumptive diagnosis of suspected viral infection.

A Peptide Antagonist of Thrombospondin-1 Promotes Abdominal Aortic Aneurysm Progression in the Angiotensin II–Infused Apolipoprotein-E–Deficient Mouse

Objective—Interaction of the activating sequence in thrombospondin-1 (TSP-1) with the conserved sequence (leucine-serine-lysine-leucine [LSKL]) in the latency-associated peptide region of latent transforming growth factor (TGF)-&bgr; complex is important in regulating TGF-&bgr;1 activity. We aimed to assess the effect of blocking peptide LSKL on the progression of pre-established abdominal aortic aneurysm in angiotensin II–infused apolipoprotein E-deficient (ApoE−/−) mice. Approach and Results—Abdominal aortic aneurysm was established in 3-month-old male ApoE−/− mice with subcutaneous infusion of angiotensin II for 28 days. After this, mice received LSKL peptide or control SLLK (serine–leucine–leucine–lysine) peptide (4 mg/kg) via daily intraperitoneal injection for an additional 2 weeks. Administration of LSKL peptide promoted larger suprarenal aortic diameter, as determined by ultrasound and morphometric analysis, and stimulated more severe atherosclerosis within the aortic arch. In addition, mice receiving LSKL peptide exhibited elevated circulating proinflammatory cytokine levels and greater inflammatory cells within the suprarenal aorta compared with controls. Mice receiving LSKL peptide showed low plasma TGF-&bgr;1 activity and low levels of aortic tissue phosphorylated to total Smad2/3. Aortic gene expression of TGF-&bgr; receptor 1 (TGFBRI) and receptor 2 (TGFBRII), but not TGF-&bgr;1 and thrombospondin-1, were lower in mice receiving LSKL peptide than controls. LSKL peptide administration was associated with greater aortic elastin fragmentation and lower expression and activity of the TGF-&bgr;1-target gene lysyl oxidase like 1 (LOXL1). Conclusions—Attenuation of thrombospondin-1-directed activation of TGF-&bgr;1 promotes abdominal aortic aneurysm and atherosclerosis progression in the angiotensin II–infused ApoE−/− mouse model.

Everolimus Limits Aortic Aneurysm in the Apolipoprotein E–Deficient Mouse by Downregulating C-C Chemokine Receptor 2 Positive Monocytes

Objective—We aimed to determine the effect of mechanistic target of rapamycin inhibitor everolimus on abdominal aortic aneurysm within the angiotensin II (A2)-infused apolipoprotein E–deficient mouse model. Approach and Results—Abdominal aortic aneurysm was induced via subcutaneous infusion of A2. Flow cytometry demonstrated increased circulating and aortic C-C chemokine receptor 2 (CCR2) monocytes during A2 infusion. The number of CCR2 monocytes present within the aorta was positively correlated with suprarenal aortic diameter. Simultaneous infusion of everolimus via a second subcutaneous osmotic micropump inhibited A2-induced aortic dilatation. Using flow cytometry and Western blot analysis, decreased aortic dilatation was associated with reduced development of CCR2 bone marrow monocytes, fewer numbers of circulating CCR2 monocytes, and lower aortic CCR2 concentration. In vitro, everolimus inhibited A2-stimulated production of interferon (IFN)-&ggr; and IFN&ggr;-induced CCR2 expression in apolipoprotein E–deficient mouse bone marrow monocytes. Further, everolimus diminished IFN&ggr;/lipopolysaccharide-stimulated M1 polarization in apolipoprotein E–deficient mouse bone marrow monocyte–differentiated macrophages. Conclusions—Systemic administration of everolimus limits aortic aneurysm in the A2-infused apolipoprotein E–deficient mouse model via suppressed development of bone marrow CCR2 monocytes and reduced egress of these cells into the circulation.

Osteoprotegerin Deficiency Limits Angiotensin II–Induced Aortic Dilatation and Rupture in the Apolipoprotein E–Knockout Mouse

Objective— Mounting evidence links osteoprotegerin with cardiovascular disease. Elevated serum and aortic tissue osteoprotegerin are associated with the presence and growth of abdominal aortic aneurysm in humans; however, a role for osteoprotegerin in abdominal aortic aneurysm pathogenesis remains to be shown. We examined the functional significance of osteoprotegerin in aortic aneurysm using an Opg-deficient mouse model and in vitro investigations. Approach and Results— Homozygous deletion of Opg in apolipoprotein E–deficient mice (ApoE −/− Opg −/−) inhibited angiotensin II-induced aortic dilatation. Survival free from aortic rupture was increased from 67% in ApoE −/− Opg +/+ controls to 94% in ApoE −/− Opg −/− mice (P=0.040). Serum concentrations of proinflammatory cytokines/chemokines, and aortic expression for cathepsin S (CTSS), matrix metalloproteinase 2, and matrix metalloproteinase 9 after 7 days (early-phase) of angiotensin II infusion were significantly reduced in ApoE −/− Opg −/− mice compared with ApoE −/− Opg +/+ controls. In addition, aortic expression of markers for an inflammatory phenotype in aortic vascular smooth muscle cells in response to early-phase of angiotensin II infusion was significantly lower in Opg-deficient mice. In vitro, human abdominal aortic aneurysm vascular smooth muscle cells produced more CTSS and exhibited increased CTSS-derived elastolytic activity than healthy aortic vascular smooth muscle cells, whereas recombinant human osteoprotegerin stimulated CTSS-dependent elastase activity in aortic vascular smooth muscle cells. Conclusions— These findings support a role for osteoprotegerin in aortic aneurysm through upregulation of CTSS, matrix metalloproteinase 2, and matrix metalloproteinase 9 within the aorta, promoting an inflammatory phenotype in aortic vascular smooth muscle cells in response to angiotensin II.

Wnt Signaling Pathway Inhibitor Sclerostin Inhibits Angiotensin II-Induced Aortic Aneurysm and Atherosclerosis.

Objective— Sclerostin (SOST) has been identified as an important regulator of bone formation; however, it has not been previously implicated in arterial disease. The aim of this study was to assess the role of SOST in aortic aneurysm (AA) and atherosclerosis using human samples, a mouse model, and in vitro investigations. Approach and Results— SOST protein was downregulated in human and mouse AA samples compared with controls. Transgenic introduction of human SOST in apolipoprotein E–deficient (ApoE−/−) mice (SOSTTg.ApoE−/−) and administration of recombinant mouse Sost inhibited angiotensin II–induced AA and atherosclerosis. Serum concentrations of several proinflammatory cytokines were significantly reduced in SOSTTg.ApoE−/− mice. Compared with controls, the aortas of mice receiving recombinant mouse Sost and SOSTTg.ApoE−/− mice showed reduced matrix degradation, reduced elastin breaks, and preserved collagen. Decreased inflammatory cell infiltration and a reduction in the expression of wingless-type mouse mammary virus integration site/&bgr;-catenin responsive genes, including matrix metalloproteinase-9, osteoprotegerin, and osteopontin, were observed in the aortas of SOSTTg.ApoE−/− mice. SOST expression was downregulated and the wingless-type mouse mammary virus integration site/&bgr;-catenin pathway was activated in human AA samples. The cytosine–phosphate–guanine islands in the SOST gene promoter showed significantly higher methylation in human AA samples compared with controls. Incubation of vascular smooth muscle cells with the demethylating agent 5-azacytidine resulted in upregulation of SOST, suggesting that SOST is epigenetically regulated. Conclusions— This study identifies that SOST is expressed in the aorta and downregulated in human AA possibly because of epigenetic silencing. Upregulating SOST inhibits AA and atherosclerosis development, with potential important implications for treating these vascular diseases.

Role of SLAM in NKT Cell Development Revealed by Transgenic Complementation in NOD Mice

Allelic variation of SLAM expression on CD4+CD8+ thymocytes has been proposed to play a major role in NKT cell development. In this article, this hypothesis is tested by the production of subcongenic mouse strains and Slamf1 transgenic lines. The long isoform of the C57BL/6 allele of Slamf1 was transgenically expressed on CD4+CD8+ thymocytes under control of an hCD2 minigene. NOD.Nkrp1b.Tg(Slamf1)1 mice, which had a 2-fold increase in SLAM protein expression on CD4+CD8+ thymocytes, had a 2-fold increase in numbers of thymic NKT cells. The additional thymic NKT cells in NOD.Nkrp1b.Tg(Slamf1)1 mice were relatively immature, with a similar subset distribution to those of congenic NOD.Nkrp1b.Nkt1 and NOD.Nkrp1b.Slamf1 mice, which also express increased levels of SLAM on CD4+CD8+ thymocytes and produce larger numbers of NKT cells. Transgenic enhancement of SLAM expression also increased IL-4 and IL-17 production in response to TCR-mediated stimulation. Paradoxically, NOD.Nkrp1b.Tg(Slamf1)2 mice, which had a 7-fold increase in SLAM expression, showed no significant increase in NKT cells numbers; on the contrary, at high transgene copy number, SLAM expression levels correlated inversely with NKT cell numbers, consistent with a contribution to negative selection. These data confirm a role for SLAM in controlling NKT cell development and are consistent with a role in both positive and negative thymic selection of NKT cells.

Modulation of Kinin B2 receptor signaling controls aortic dilatation and rupture in the Angiotensin II-infused apolipoprotein E-deficient mouse

Objective—Abdominal aortic aneurysm (AAA) is an important cause of mortality in older adults. Activity of the local kallikrein–kinin system may be important in cardiovascular disease. The effect of kinin B2 receptor (B2R) agonist and antagonist peptides on experimental AAA was investigated. Approach and Results—AAA was induced in apolipoprotein E–deficient mice via infusion of angiotensin II (1.0 &mgr;g/kg per minute SC). B2R agonists or antagonists were given via injection (2 mg/kg IP) every other day. The B2R agonist (B9772) promoted aortic rupture in response to angiotensin II associated with an increase in neutrophil infiltration of the aorta in comparison to controls. Mice receiving a B2R/kinin B1 receptor antagonist (B9430) were relatively protected from aortic rupture. Neutrophil depletion abrogated the ability of the B2R agonist to promote aortic rupture. Progression of angiotensin II–induced aortic dilatation was inhibited in mice receiving a B2R antagonist (B9330). Secretion of metalloproteinase-2 and -9, osteoprotegerin, and osteopontin by human AAA explant was reduced in the presence of the B2R antagonist (B9330). B2R agonist and antagonist peptides enhanced and inhibited, respectively, angiotensin II–induced neutrophil activation and aortic smooth muscle cell inflammatory phenotype. The B2R antagonist (B9330; 5 &mgr;g) delivered directly to the aortic wall 1 week post-AAA induction with calcium phosphate in a rat model reduced aneurysm growth associated with downregulation of aortic metalloproteinase-9. Conclusions—B2R signaling promotes aortic rupture within a mouse model associated with the ability to stimulate inflammatory phenotypes of neutrophils and vascular smooth muscle cells. B2R antagonism could be a potential therapy for AAA.

Parenteral administration of factor Xa/IIa inhibitors limits experimental aortic aneurysm and atherosclerosis

Intraluminal thrombus is a consistent feature of human abdominal aortic aneurysm (AAA). Coagulation factor Xa (FXa) catalyses FII to thrombin (FIIa). We examined the effect of FXa/FIIa inhibition on experimental aortic aneurysm in apolipoprotein E-deficient (ApoE−/−) mice infused with angiotensin II (AngII). The concentration of FXa within the supra-renal aorta (SRA) correlated positively with SRA diameter. Parenteral administration of enoxaparin (FXa/IIa inhibitor) and fondaparinux (FXa inhibitor) over 14 days reduced to severity of aortic aneurysm and atherosclerosis in AngII-infused ApoE−/− mice. Enteral administration of the FIIa inhibitor dabigatran had no significant effect. Aortic protease-activated receptor (PAR)-2 expression increased in response to AngII infusion. Fondaparinux reduced SRA levels of FXa, FIIa, PAR-2, matrix metalloproteinase (MMP)2, Smad2/3 phosphorylation, and MOMA-2 positive cells in the mouse model. FXa stimulated Smad2/3 phosphorylation and MMP2 expression in aortic vascular smooth muscle cells (VSMC) in vitro. Expression of MMP2 in FXa-stimulated VSMC was downregulated in the presence of a PAR-2 but not a PAR-1 inhibitor. These findings suggest that FXa/FIIa inhibition limits aortic aneurysm and atherosclerosis severity due to down-regulation of vascular PAR-2-mediated Smad2/3 signalling and MMP2 expression. Inhibition of FXa/FIIa may be a potential therapy for limiting aortic aneurysm.

High serum thrombospondin-1 concentration is associated with slower abdominal aortic aneurysm growth and deficiency promotes angiotensin-II induced aneurysm in mice

Abdominal aortic aneurysm (AAA) is a common age-related vascular disease characterized by progressive weakening and dilatation of the aortic wall. Thrombospondin-1 (TSP-1; gene Thbs1) is a member of the matricellular protein family important in the control of extracellular matrix (ECM) remodelling. In the present study, the association of serum TSP-1 concentration with AAA progression was assessed in 276 men that underwent repeated ultrasound for a median 5.5 years. AAA growth was negatively correlated with serum TSP-1 concentration (Spearmans rho -0.129, P=0.033). Men with TSP-1 in the highest quartile had a reduced likelihood of AAA growth greater than median during follow-up (OR: 0.40; 95% confidence interval (CI): 0.19-0.84, P=0.016, adjusted for other risk factors). Immunohistochemical staining for TSP-1 was reduced in AAA body tissues compared with the relatively normal AAA neck. To further assess the role of TSP-1 in AAA initiation and progression, combined TSP-1 and apolipoprotein deficient (Thbs1-/-ApoE-/-, n=20) and control mice (ApoE-/-, n=20) were infused subcutaneously with angiotensin II (AngII) for 28 days. Following AngII infusion, Thbs1-/- ApoE-/- mice had larger AAAs by ultrasound (P=0.024) and ex vivo morphometry measurement (P=0.006). The Thbs1-/-ApoE-/- mice also showed increased elastin filament degradation along with elevated systemic levels and aortic expression of matrix metalloproteinase (MMP)-9. Suprarenal aortic segments and vascular smooth muscle cells (VSMCs) isolated from Thbs1-/-ApoE-/- mice showed reduced collagen 3A1 gene expression. Furthermore, Thbs1-/-ApoE-/- mice had reduced aortic expression of low-density lipoprotein (LDL) receptor-related protein 1. Collectively, findings from the present study suggest that TSP-1 deficiency promotes maladaptive remodelling of the ECM leading to accelerated AAA progression.

Influence of apolipoprotein E, age and aortic site on calcium phosphate induced abdominal aortic aneurysm in mice

OBJECTIVE To assess relevant features of abdominal aortic aneurysms (AAA) induced by calcium phosphate within a mouse model. Specifically we investigated: (1) whether apolipoprotein E deficiency and older age promoted AAA formation, and (2) whether the local application of calcium phosphate affected the size of distant aortic segments. METHODS AAA was induced by application of calcium phosphate to the infra-renal aortas of 3 and 7 month old male mice. AAA induction was assessed by calculating expansion of the infra-renal aortic diameter over 1-4 weeks. Aortic samples were assessed to quantify calcification, macrophages infiltration, elastic lamellar degradation and apoptosis. Blood pressure was measured by the tail cuff method, and plasma concentrations of total cholesterol, low density lipoprotein and very low density lipoprotein cholesterol, and pro-inflammatory cytokines were measured using commercially available kits. The maximum diameters of the aortic arch, thoracic and supra-renal aorta at sacrifice were measured by morphometry and the mean maximal diameter of these three aortic segments was calculated. RESULTS The median expansion of the infra-renal aorta 2 weeks after AAA induction was significantly greater in apolipoprotein E deficient (ApoE(-/-)) mice than in age- and gender-matched wild type controls [275.8% (IQR 193.8%-348.5%) versus 94.7% (IQR 47.8%-163.4%), P = 0.02]. The greater aortic expansion in ApoE(-/-) mice was associated with aortic calcification, macrophage infiltration, elastic lamellar degradation and apoptosis of cells in the media and adventitia. The plasma low density lipoprotein/very low density lipoprotein cholesterol concentrations 2 weeks after AAA induction were positively correlated with the expansion of the infra-renal aorta induced by calcium phosphate. The median expansion of the infra-renal aorta 2 weeks after AAA induction was similar in 3 and 7 month old wild type mice. The local administration of calcium phosphate was associated with an increase in the mean maximal diameter of distant aortic segments, but not associated with changes in the concentrations of pro-inflammatory markers in either the plasma or the spleen. CONCLUSION This study suggests that apolipoprotein E deficiency, but not age, predisposes to AAA induced within the calcium phosphate model. Increased AAA expansion in ApoE(-/-) mice was associated with calcification, macrophage infiltration, elastic lamellar degradation, and cell apoptosis. Local application of calcium phosphate also promoted dilation of distant aortic segments.