Hanna M. Björck

Effects of PDGF-C and PDGF-D on monocyte migration and MMP-2 and MMP-9 expression

BACKGROUND AND AIMS Atherosclerosis is a chronic inflammatory process involving the activity of several cytokines and growth factors. Platelet-derived growth factor-A (PDGF-A) and PDGF-B are important mitogens and chemoattractants for monocytes as well as smooth muscle cells. We sought to identify the role of PDGF-C and PDGF-D, two new members of the PDGF family, in monocyte migration and differentiation. We also assessed their effects in regulating matrix metalloproteinase-2 (MMP-2) and MMP-9, which are important for cell migration. METHODS AND RESULTS PDGF-C and PDGF-D were expressed in macrophages, smooth muscle cells, and endothelial cells in human atherosclerotic plaques, as shown by immunohistochemical analysis. PDGF-C and PDGF-D mRNA and protein expression was induced after differentiation of THP-1 monocytes to macrophages, and both PDGF-C and PDGF-D induced MMP-9 mRNA expression in a concentration-dependent manner. Treatment of cells with PDGF-C or PDGF-D enhanced the secretion of MMP-2 and MMP-9 in a cell-dependent manner. In a migration assay using a Boyden chamber with 8 microm pore size, PDGF-C and PDGF-D attracted THP-1 monocytes in a concentration-dependent manner. CONCLUSIONS Our data suggest that PDGF-C and PDGF-D, like PDGF-A and PDGF-B, play important roles in atherosclerosis by stimulating MMP activity and influencing monocyte migration.

Association of genetic variation on chromosome 9p21.3 and arterial stiffness

Objectives.  Genome wide association studies have consistently reported associations between a region on chromosome 9p21.3 and a broad range of vascular diseases, such as coronary artery disease (CAD), aortic and intracranial aneurysms and type‐2 diabetes (T2D). However, clear associations with intermediate phenotypes have not been described so far. To shed light on a possible influence of this chromosomal region on arterial wall integrity, we analysed associations between single nucleotide polymorphisms (SNPs) and degree of stiffness of the abdominal aorta in elderly individuals.

The association between circulating angiotensin-converting enzyme and cardiovascular risk in the elderly: a cross-sectional study

Introduction: A polymorphism in the angiotensin-converting enzyme gene (ACE I/D polymorphism) has been associated with increased risk for cardiovascular disease (CVD). This polymorphism affects the level of circulating ACE, but there is great individual variation, even between those with the same genotype. Few previous studies have investigated the link between circulating ACE and cardiovascular risk. The aim of this study was to investigate this association, and to examine the relationship between ACE level, ACE genotype and CVD. Materials and methods : The study population consisted of 322 men and 350 women aged 69—87. Plasma ACE level was determined using enzyme-linked immunosorbent assay (ELISA), and ACE genotype was analysed using PCR followed by gel electrophoresis. Results: In men, ACE levels increased with increasing number of cardiovascular risk factors (p = 0.003). There was a significant association in men between increased ACE level and both diabetes (p = 0.007) and smoking (p = 0.037). Conclusions:This study shows that cardiovascular risk factors (such as smoking and diabetes) are associated with higher levels of circulating ACE in men. High ACE levels may represent one of the cellular mechanisms involved in producing the vascular damage associated with cardiovascular risk factors.

Aneurysm Development in Patients With a Bicuspid Aortic Valve Is Not Associated With Transforming Growth Factor-β Activation

Objective— Patients with bicuspid aortic valve (BAV) have an increased risk of developing ascending aortic aneurysms. Transforming growth factor-&bgr; (TGF&bgr;) is a crucial factor of vascular remodeling, the impaired signaling of which can alter the structure and composition of the extracellular matrix. In this study, we analyzed the activity of TGF&bgr; in aneurysmal and nonaneurysmal ascending aorta from BAV patients, using tricuspid aortic valve (TAV) patients as a reference group. Approach and Results— The response to exogenous TGF&bgr; was analyzed with regard to gene expression in primary aortic smooth muscle cells that were isolated from 7 BAV and 5 TAV patients and in valve fibroblasts from 7 BAV and 8 TAV patients. The set of genes that were significantly changed by TGF&bgr; (217 genes) was compared with gene expression profiles of the ascending aorta from BAV and TAV patients (139 arrays). By principle component analysis, based on the 217 genes, gene expression differed significantly in the intima/media region between aneurysmal BAV and TAV aortas, driven by the response in TAV patients. During aneurysm development the levels of phosphorylated SMADs and the availability of free TGF&bgr; were lower in BAV patients compared with TAV. Confocal microscopy analysis showed a higher colocalization of latency associated peptide and latent TGF&bgr; binding protein 3 in BAV aortas. Conclusions— Our findings suggest that TGF&bgr; activation during aneurysm formation is muted in patients with BAV, possibly as a result of an increased TGF&bgr; sequestration in the extracellular space.

Identification of a novel flow-mediated gene expression signature in patients with bicuspid aortic valve

Individuals with bicuspid aortic valve (BAV) are at significantly higher risk of developing serious aortic complications than individuals with tricuspid aortic valves (TAV). Studies have indicated an altered aortic blood flow in patients with BAV; however, the extent to which altered flow influences the pathological state of BAV aorta is unclear. In the present study, we dissected flow-mediated aortic gene expression in patients undergoing elective open heart surgery. A large collection of public microarray data sets were firstly screened for consistent co-expression with five well-characterized flow-regulated genes (query genes). Genes with co-expression probability of >0.5 were selected and further analysed in expression profiles (127 arrays) from ascending aorta of BAV and TAV patients. Forty-four genes satisfied two filtering criteria: a significant correlation with one or more of the query genes (R > 0.40) and differential expression between patients with BAV and TAV. No gene fulfilled the criteria in mammary artery (88 arrays), an artery not in direct contact with the valve. Fifty-five percent of the genes significantly altered between BAV and TAV patients showed differential expression between two identified flow regions in the rat aorta. A large proportion of the identified genes were related to angiogenesis and/or wound healing, with pro-angiogenesis genes downregulated and inhibitory genes upregulated in patients with BAV. Moreover, differential expression of ZFP36, GRP116 and PKD2 was confirmed using immunohistochemistry. Implementing a new strategy, we have demonstrated an angiostatic gene expression signature in patients with BAV, indicating impaired wound healing in these patients, potentially involved in BAV-associated aortopathy.

Mesenchymal state of intimal cells may explain higher propensity to ascending aortic aneurysm in bicuspid aortic valves

Individuals with a bicuspid aortic valve (BAV) are at significantly higher risk of developing aortic complications than individuals with tricuspid aortic valves (TAV) and defective signaling during the embryonic development and/or life time exposure to abnormal hemodynamic have been proposed as underlying factors. However, an explanation for the molecular mechanisms of aortopathy in BAV has not yet been provided. We combined proteomics, RNA analyses, immunohistochemistry, and electron microscopy to identify molecular differences in samples of non-dilated ascending aortas from BAV (N = 62) and TAV (N = 54) patients. Proteomic analysis was also performed for dilated aortas (N = 6 BAV and N = 5 TAV) to gain further insight into the aortopathy of BAV. Our results collectively showed the molecular signature of an endothelial/epithelial-mesenchymal (EndMT/EMT) transition-like process, associated with instability of intimal cell junctions and activation of RHOA pathway in the intima and media layers of ascending aorta in BAV patients. We propose that an improper regulation of EndMT/EMT during the spatiotemporally related embryogenesis of semilunar valves and ascending aorta in BAV individuals may result in aortic immaturity and instability prior to dilation. Exasperation of EndMT/EMT state in post embryonic life and/or exposure to non-physiological hemodynamic could lead to the aneurysm of ascending aorta in BAV individuals.

Characterization of shear-sensitive genes in the normal rat aorta identifies Hand2 as a major flow-responsive transcription factor.

Objective Shear forces play a key role in the maintenance of vessel wall integrity. Current understanding regarding shear-dependent gene expression is mainly based on in vitro or in vivo observations with experimentally deranged shear, hence reflecting acute molecular events in relation to flow. Our objective was to combine computational fluid dynamic (CFD) simulations with global microarray analysis to study flow-dependent vessel wall biology in the aortic wall under physiological conditions. Methods and Results Male Wistar rats were used. Animal-specific wall shear stress (WSS) magnitude and vector direction were estimated using CFD based on aortic geometry and flow information acquired by magnetic resonance imaging. Two distinct flow pattern regions were identified in the normal rat aortic arch; the distal part of the lesser curvature being exposed to low WSS and a non-uniform vector direction, and a region along the greater curvature being subjected to markedly higher levels of WSS and a uniform vector direction. Microarray analysis identified numerous novel mechanosensitive genes, including Trpc4 and Fgf12, and confirmed well-known ones, e.g. Klf2 and Nrf2. Gene ontology analysis revealed an over-representation of genes involved in transcriptional regulation. The most differentially expressed gene, Hand2, is a transcription factor previously shown to be involved in extracellular matrix remodeling. HAND2 protein was endothelial specific and showed higher expression in the regions exposed to low WSS with disturbed flow. Conclusions Microarray analysis validated the CFD-defined WSS regions in the rat aortic arch, and identified numerous novel shear-sensitive genes. Defining the functional importance of these genes in relation to atherosusceptibility may provide important insight into the understanding of vascular pathology.

Impaired Abdominal aortic wall Integrity in Elderly Men Carrying the Angiotensin-converting Enzyme D Allele

OBJECTIVE A polymorphism in the angiotensin-converting-enzyme gene (ACE I/D) has been associated with abdominal aortic aneurysm and a link between aortic aneurysm and aortic stiffness has been suggested. This study aimed to explore the links between ACE I/D polymorphism, circulating ACE and abdominal aortic wall integrity as reflected by abdominal aortic wall stiffness. MATERIAL A total of 212 men and 194 women, aged 70-88 years, were studied. METHODS Mechanical properties of the abdominal aorta were determined using the Wall Track System, ACE genotype using the polymerase chain reaction (PCR) and circulating ACE level by enzyme-linked immunosorbent assay (ELISA). RESULTS In men, pulsatile diameter change differed between genotypes (II 0.70, ID 0.55 and DD 0.60 mm, P = 0.048), whereas a tendency was seen for distensibility coefficient (DC) (II 10.38, ID 7.68 and ID 8.79, P = 0.058). Using a dominant model (II vs. ID/DD), men carrying the ACE D allele had lower pulsatile diameter change (P = 0.014) and DC (P = 0.017) than II carriers. Multiple regression analyses showed additional associations between the D allele and increased stiffness β, and reduced compliance coefficient. CONCLUSION Men carrying the ACE D allele have stiffer abdominal aortas compared with II carriers. Deranged abdominal aortic stiffness indicates impaired vessel wall integrity, which, along with other local predisposing factors, may be important in aneurysmal disease.

Gender-Specific Association of the Plasminogen Activator Inhibitor-1 4G/5G Polymorphism With Central Arterial Blood Pressure

BACKGROUND The functional plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphism has previously been associated with hypertension. In recent years, central blood pressure, rather than brachial has been argued a better measure of cardiovascular damage and clinical outcome. The aim of this study was to investigate the possible influence of the 4G/5G polymorphism on central arterial blood pressure in a cohort of elderly individuals. METHODS We studied 410 individuals, 216 men and 194 women, aged 70-88. Central pressures and pulse waveforms were calculated from the radial artery pressure waveform by the use of the SphygmoCor system and a generalized transfer function. Brachial pressure was recorded using oscillometric technique (Dinamap, Critikon, Tampa, FL). PAI-1 antigen was determined in plasma. RESULTS The results showed that central pressures were higher in women carrying the PAI-1 4G/4G genotype compared to female carriers of the 5G/5G genotype, (P = 0.025, P = 0.002, and P = 0.002 for central systolic-, diastolic-, and mean arterial pressure, respectively). The association remained after adjustment for potentially confounding factors related to hypertension. No association of the PAI-1 genotype with blood pressure was found in men. Multiple regression analysis revealed an association between PAI-1 genotype and plasma PAI-1 levels (P = 0.048). CONCLUSIONS Our findings show a gender-specific association of the PAI-1 4G/5G polymorphism with central arterial blood pressure. The genotype effect was independent of other risk factors related to hypertension, suggesting that impaired fibrinolytic potential may play an important role in the development of central hypertension in women.

Aneurysm Development in Patients With Bicuspid Aortic Valve (BAV): Possible Connection to Repair Deficiency?

Thoracic aortic aneurysm (TAA) is manifested by progressive enlargement of the thoracic aorta due to destructive changes in the connective tissue of the media and adventitia in the aortic wall. This process, which is also known as cystic medial degeneration, may ultimately lead to aortic dissection or rupture [1, 2]. TAAs are characterized by extensive loss of smooth muscle cells (SMCs) and disruption of elastin and collagen but, unlike abdominal aortic aneurysms, are not associated with intimal atherosclerosis and chronic inflammation [3,4]. Since therapeutic agents that may halt or reverse the process of aortic wall deterioration are absent, the only available therapeutic recommendation is elective surgical intervention. TAA may occur in the presence of a tricuspid or a bicuspid aortic valve (TAV and BAV), respectively. The pathogenesis of TAA is complex, involving multiple interacting processes, and in this review, we will focus on the latest findings in our laboratory and others, which implies that in spite of phenotypic similarities of the end point of aneurysm, the process of cystic medial degeneration may indeed occur by fundamentally different mechanisms in BAV and TAV patients. Ascending aortic dilatation occurs more frequently and at a younger age in patients with BAV than TAV. It has been estimated that 50%–70% of individuals with BAV develop aortic dilatation. Furthermore, a higher proportion of BAV patients develop other cardiac complications such as aortic valve stenosis and aortic regurgitation [5,6].