Maria Nataatmadja

Abnormal Extracellular Matrix Protein Transport Associated With Increased Apoptosis of Vascular Smooth Muscle Cells in Marfan Syndrome and Bicuspid Aortic Valve Thoracic Aortic Aneurysm

Background—Marfan syndrome (MS) is a genetic disorder caused by a mutation in the fibrillin gene FBN1. Bicuspid aortic valve (BAV) is a congenital heart malformation of unknown cause. Both conditions are associated with ascending aortic aneurysm and premature death. This study examined the relationship among the secretion of extracellular matrix proteins fibrillin, fibronectin, tenascin, and vascular smooth muscle cell (VSMC) apoptosis. The role of matrix metalloproteinase (MMP)-2 in VSMC apoptosis was studied in MS aneurysm. Methods and Results—Aneurysm tissue was obtained from patients undergoing surgery (MS: 4 M, 1 F, age 27–45 years; BAV: 3 M, 2 F, age 28–65 years). Normal aorta from subjects with nonaneurysm disease was also collected (4 M, 1 F, age 23–93 years). MS and BAV aneurysm histology showed areas of cystic medial necrosis (CMN) without inflammatory infiltrate. Immunohistochemical study of cultured MS and BAV VSMC showed intracellular accumulation and reduction of extracellular distribution of fibrillin, fibronectin, and tenascin. Western blot showed no increase in expression of fibrillin, fibronectin, or tenascin in MS or BAV VSMC and increased expression of MMP-2 in MS VSMCs.There was 4-fold increase in loss of cultured VSMC incubated in serum-free medium for 24 hours in both MS (27±8%) and BAV (32±14%) compared with control (7±5%). Conclusions—In MS and BAV there is alteration in both the amount and quality of secreted proteins and an increased degree of VSMC apoptosis. Up-regulation of MMP-2 might play a role in VSMC apoptosis in MS VSMC. The findings suggest the presence of a fundamental cellular abnormality in BAV thoracic aorta, possibly of genetic origin.

Collagen in the scarless fetal skin wound: Detection with Picrosirius-polarization

Our group has developed an ovine model of deep dermal, partial‐thickness burn where the fetus heals scarlessly and the lamb heals with scar. The comparison of collagen structure between these two different mechanisms of healing may elucidate the process of scarless wound healing. Picrosirius staining followed by polarized light microscopy was used to visualize collagen fibers, with digital capture and analysis. Collagen deposition increased with fetal age and the fibers became thicker, changing from green (type III collagen) to yellow/red (type I collagen). The ratio of type III collagen to type I was high in the fetus (166), whereas the lamb had a much lower ratio (0.2). After burn, the ratios of type III to type I collagen did not differ from those in control skin for either fetus or lamb. The fetal tissue maintained normal tissue architecture after burn while the lamb tissue showed irregular collagen organization. In conclusion, the type or amount of collagen does not alter significantly after injury. Tissue architecture differed between fetal and lamb tissue, suggesting that scar development is related to collagen cross‐linking or arrangement. This study indicates that healing in the scarless fetal wound is representative of the normal fetal growth pattern, rather than a “response” to burn injury.

Overexpression of Transforming Growth Factor-β Is Associated With Increased Hyaluronan Content and Impairment of Repair in Marfan Syndrome Aortic Aneurysm

Background— Marfan syndrome (MFS), a condition caused by fibrillin-1 gene mutation is associated with aortic aneurysm that shows elastic lamellae disruption, accumulation of glycosaminoglycans, and vascular smooth muscle cell (VSMC) apoptosis with minimal inflammatory response. We examined aneurysm tissue and cultured cells for expression of transforming growth factor-beta1 to -beta3 (TGFβ1 to 3), hyaluronan content, apoptosis, markers of cell migration, and infiltration of vascular progenitor cells (CD34). Methods and Results— MFS aortic aneurysm (6 males, 5 females; age 8 to 78 years) and normal aorta (5 males, 3 females; age 22 to 56 years) were used. Immunohistochemistry showed increased expression of TGFβ1 to 3, hyaluronan, and CD34-positive microcapillaries in MFS aneurysm compared with control. There was increased expression of TGFβ1 to 3 and hyaluronan in MFS cultured VSMCs, adventitial fibroblasts (AF), and skin fibroblasts (SF). Apoptosis was increased in MFS (VSMC: mean cell loss in MFS 29%, n of subjects=5, versus control 8%, n=3, P<0.05; AF: 28%, n=5 versus 7%, n=5, P<0.05; SF: 29%, n=3 versus 4%, n=3, not significant). In MFS, there was a 2-fold increase in adventitial microcapillaries containing CD34-positive cells compared with control tissue. Scratch wound assay showed absence of CD44, MT1-MMP, and β-3 integrin at the leading edge of migration in MFS indicating altered directional migration. Western blot showed increased expression of TGFβ1 to 3 in MFS but no change in expression of CD44, MT1-MMP, or β-3 integrin compared with controls. Conclusions— There was overexpression of TGF-β in MFS associated with altered hyaluronan synthesis, increased apoptosis, impaired progenitor cell recruitment, and abnormal directional migration. These factors limit tissue repair and are likely to contribute to aneurysm development.

The value of screening in siblings of patients with abdominal aortic aneurysm.

OBJECTIVES This study aimed to determine the incidence of abdominal aortic aneurysm (AAA) in a large group of siblings of Australian AAA patients to determine if screening in this group is justified. METHODS 1254 siblings of 400 index AAA patients were identified and offered aortic ultrasound screening. An age and sex matched control group was recruited from patients having abdominal CT scans for non-vascular indications. AAA was defined by an infrarenal aortic diameter of > or =3 cm or a ratio of the infrarenal to suprarenal aortic diameter of > or =2.0. A ratio of 1.0-1.5 was considered normal, and a ratio of >1.5 to <2.0 was considered ectatic. Aortic enlargement was defined as ectasia or aneurysm. RESULTS 276 (22%) siblings could be contacted and agreed to screening or had previously been diagnosed with AAA. All 118 controls had normal diameter aortas. 55/276 siblings had previously been diagnosed with AAA. The remaining 221 siblings underwent ultrasound screening. Overall, 30% (84/276) had enlarged aortas (5% ectasia, 25% aneurysmal); 43% of male siblings (64/150) and 16% of females siblings (20/126). The incidence was 45% in brothers of female index patients, 42% in brothers of male patients, 23% in sisters of female patients, and 14% in sisters of male index patients. CONCLUSIONS The overall incidence of aortic enlargement of 30% found in this study warrants a targeted screening approach with ultrasound for all siblings of patients with AAA. A similar targeted approach for screening of the children of AAA patients would also seem advisable.

Selection of reference genes for normalisation of real-time RT-PCR in brain-stem death injury in Ovis aries

BackgroundHeart and lung transplantation is frequently the only therapeutic option for patients with end stage cardio respiratory disease. Organ donation post brain stem death (BSD) is a pre-requisite, yet BSD itself causes such severe damage that many organs offered for donation are unusable, with lung being the organ most affected by BSD. In Australia and New Zealand, less than 50% of lungs offered for donation post BSD are suitable for transplantation, as compared with over 90% of kidneys, resulting in patients dying for lack of suitable lungs. Our group has developed a novel 24 h sheep BSD model to mimic the physiological milieu of the typical human organ donor. Characterisation of the gene expression changes associated with BSD is critical and will assist in determining the aetiology of lung damage post BSD. Real-time PCR is a highly sensitive method involving multiple steps from extraction to processing RNA so the choice of housekeeping genes is important in obtaining reliable results. Little information however, is available on the expression stability of reference genes in the sheep pulmonary artery and lung. We aimed to establish a set of stably expressed reference genes for use as a standard for analysis of gene expression changes in BSD.ResultsWe evaluated the expression stability of 6 candidate normalisation genes (ACTB, GAPDH, HGPRT, PGK1, PPIA and RPLP0) using real time quantitative PCR. There was a wide range of Ct-values within each tissue for pulmonary artery (15–24) and lung (16–25) but the expression pattern for each gene was similar across the two tissues. After geNorm analysis, ACTB and PPIA were shown to be the most stably expressed in the pulmonary artery and ACTB and PGK1 in the lung tissue of BSD sheep.ConclusionAccurate normalisation is critical in obtaining reliable and reproducible results in gene expression studies. This study demonstrates tissue associated variability in the selection of these normalisation genes in BSD sheep and underlines the importance of selecting the correct reference genes for both the animal model and tissue studied.

Downregulation of transforming growth factor, beta receptor 2 and Notch signaling pathway in human abdominal aortic aneurysm

OBJECTIVE Mutations in FBN1 and TGFBR2 genes are the main causative mutations identified in Marfan syndrome (MFS). The major vascular complication of MFS is aneurysm formation. Abdominal aortic aneurysm (AAA) is an acquired disease of later life of unknown etiology. The aim of this study was to examine if genetic aberrations in MFS-related genes FBN1 and TGFBR2 are present in patients with AAA. METHODS We assessed the presence of copy number variation (CNV) in FBN1 and TGFBR2 genes in AAA biopsies from twelve patients. We also analyzed the expression of these genes in AAA biopsies compared to control biopsies from six organ donors. In addition we assessed the expression of two members of the Notch signaling pathway NOTCH3 and HEY2 as well as aortic smooth muscle cell (AoSMC) differentiation marker TAGLN in AAA and control biopsies. RESULTS Loss of one copy (deletion) of the FBN1 exon 66 sequence and TGFBR2 exon 8 was identified in 7 (58%) and 11 (92%) of the 12 AAA biopsies. No copy number amplifications (duplications) were detected. Patients carrying TGFBR2 exon 8 deletion showed marked downregulation of this gene in AAA biopsies compared to control biopsies (0.699 vs. 1.765, p = 0.038). Notch signaling components NOTCH3 and HEY2 were markedly downregulated in AAA, while expression of the AoSMC differentiation marker TAGLN did not differ between AAA and control biopsies (0.468 vs. 0.486, p = 0.546). CONCLUSION This study suggests an acquired impairment in TGF-β signaling that along with downregulation of the Notch signaling pathway may contribute to the pathogenesis of AAA.

Vascular Remodeling in the Internal Mammary Artery Graft and Association With In Situ Endothelin-1 and Receptor Expression

Background— The vasoconstricting peptide endothelin-1 (ET-1) has been associated with atherosclerotic cardiovascular disease, vascular smooth muscle cell (VSMC) growth stimulation, and intimal thickening. ET-1 binds 2 receptor subtypes, endothelin A and B, and the ETA receptor mediates vasoconstriction and VSMC growth. This study aims to quantitatively assess arterial remodeling variables and compare them with changes in ET-1, ETA, and ETB expression in the internal mammary artery (IMA). Methods and Results— Specimens from 55 coronary artery disease (CAD) patients (45 men, 10 women; mean age 65 years) and 14 control IMA specimens (from 7 men and 7 women; mean age 45 years) were collected. IMA cross sections were assessed by histochemical and immunohistochemical staining methods to quantify the levels of medionecrosis, fibrosis, VSMC growth, ET-1, ETA, ETB, and macrophage infiltration. The percentage area of medionecrosis in the patients was almost double that in the controls (31.85±14.52% versus 17.10±9.96%, P=0.0006). Total and type 1 collagen was significantly increased compared with controls (65.8±18.3% versus 33.7±13.7%, P=0.07, and 14.2±10.0% versus 4.8±2.8%, P=0.01, respectively). Despite ACE and/or statin therapy, ET-1 expression and cell cycling were significantly elevated in the patient IMAs relative to the controls (46.27±18.46 versus 8.56±8.42, P=0.0001, and 37.29±12.88 versus 11.06±8.18, P=0.0001, respectively). ETA and ETB staining was elevated in the patient vessels (46.88±11.52% versus 18.58±7.65%, P=0.0001, and 42.98±7.08% versus 34.73±5.20%, P=0.0067, respectively). A mild presence of macrophages was noted in all sections. Conclusions— Elevated distribution of collagen indicative of fibrosis coupled with increased cell cycling and high levels of ET-1 and ETA expression in the absence of chronic inflammation suggests altered IMA VSMC regulation is fundamental to the remodeling process.

Differential gene expression in the proximal neck of human abdominal aortic aneurysm

OBJECTIVE Abdominal aortic aneurysm (AAA) represents a common cause of morbidity and mortality in elderly populations but the mechanisms involved in AAA formation remain incompletely understood. Previous human studies have focused on biopsies obtained from the center of the AAA however it is likely that pathological changes also occur in relatively normal appearing aorta away from the site of main dilatation. The aim of this study was to assess the gene expression profile of biopsies obtained from the neck of human AAAs. METHODS We performed a microarray study of aortic neck specimens obtained from 14 patients with AAA and 8 control aortic specimens obtained from organ donors. Two-fold differentially expressed genes were identified with correction for multiple testing. Mechanisms represented by differentially expressed genes were identified using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Some of the differentially expressed genes were validated by quantitative real-time PCR (qPCR) and immunohistochemistry. RESULTS We identified 1047 differentially expressed genes in AAA necks. The KEGG analysis revealed marked upregulation of genes related to immunity. These pathways included cytokine-cytokine receptor interaction (P = 8.67*10(-12)), chemokine signaling pathway (P = 5.76*10(-07)), and antigen processing and presentation (P = 4.00*10(-04)). Examples of differentially expressed genes validated by qPCR included the T-cells marker CD44 (2.16-fold upregulated, P = 0.008) and the B-cells marker CD19 (3.14-fold upregulated, P = 0.029). The presence of B-cells in AAA necks was confirmed by immunohistochemistry. CONCLUSIONS The role of immunity in AAA is controversial. This study suggests that immune pathways are also upregulated within the undilated aorta proximal to an AAA.

Histopathology and fibrillin-1 distribution in severe early onset Marfan syndrome

Marfan syndrome (MFS) is an autosomal dominant condition which may involve the cardiovascular, ocular, skeletal, and other systems. Mutations causing MFS are found in the FBN1 gene, encoding fibrillin‐1, an extracellular matrix protein involved in microfibril formation. In the most severe cases, mutations are generally found in exons 24–32, and children with these mutations usually die in the first years of life, of cardiopulmonary failure. We present clinical, molecular and histopathological studies on a patient with severe early onset MFS. He has a mutation in exon 25 of FBN1, a G>A transition at nucleotide position 3131 that converts the codon TGC, coding for cysteine at position 1044, to TAC, coding for tyrosine (C1044Y). This has resulted in abnormalities of the extracellular matrix and a severe clinical phenotype, although he has survived to the age of 14 years.

Osteopontin alters endothelial and valvular interstitial cell behaviour in calcific aortic valve stenosis through HMGB1 regulation

OBJECTIVES Calcific aortic valve stenosis (CAVS) is an important clinical problem predominantly affecting elderly individuals. Studies suggest that the progression of CAVS is actively regulated with valve endothelial injury leading to inflammation, fibrosis and calcification. The aim of this study was to delineate the possible regulatory role of osteopontin (OPN) on high-mobility group box 1 (HMGB1) function and the associated inflammatory and fibrotic response in CAVS. METHODS Aortic valve leaflets were collected from CAVS patients undergoing aortic valve replacement (n = 40), and control aortic valve leaflets were obtained from heart transplant recipients (n = 15). Valves and plasma were analysed by quantitative real-time polymerase chain reaction (PCR), immunohistochemical staining and Western blot. Recombinant OPN or neutralizing OPN antibody was added to cultured endothelial and valvular interstitial cells (VICs), and cell proliferation scores and HMGB1 expression were assessed. RESULTS CAVS valves had a decreased total percentage of VICs but increased numbers of infiltrating macrophages relative to control valves. RT-PCR studies showed higher expression of OPN, the inflammatory cytokine tumour necrosis factor-alpha as well as markers of fibrosis, tissue inhibitor of matrix metalloproteinase 1 and matrix metalloproteinase 2 in CAVS valves. Elevated expression of OPN was also observed in plasma of CAVS patients compared with controls. HMGB1 was detected in the secretory granules of cultured valve endothelial and VICs derived from CAVS valves. The addition of exogenous OPN inhibited the proliferation of cultured endothelial and VICs from CAVS valves and was associated with the extracellular expression of HMGB1, whereas neutralizing OPN had the opposite effect. CONCLUSIONS We conclude that altered OPN expression in CAVS affects cellular HMGB1 function inducing cytoplasmic translocation and secretion of HMGB1 in endothelial cells and VICs, thus indicating a regulatory role for OPN in the progression of CAVS through alteration of HMGB1 function.