A Late Complication in a Surgically Corrected ALCAPA Patient.

Late complications in surgically corrected ALCAPA patients are rare. We describe an interesting case of a patient with a thrombosed giant right coronary artery aneurysm which was discovered on a chest X-ray. (Level of Difficulty: Intermediate.).

Distinct CD44 splice variants differentially affect collateral artery growth.

OBJECTIVE: Lack of the adhesion molecule CD44 reduces collateral artery growth (arteriogenesis) in a murine hindlimb model. CD44 function is influenced by expression of 10 alternatively spliced exons (v1-v10), with unknown effects on arteriogenesis. As the variant exon CD44v3 binds heparan sulphate and facilitates preservation of growth factors, we hypothesized that the variably spliced exon region of CD44, especially exon CD44v3, is involved in arteriogenesis. MATERIALS AND METHODS: The right femoral artery of C57BL/6J-mice was ligated and tissue was processed for histological and qPCR analysis of CD44-isoform expression. Microsphere perfusion measurements were performed in mice lacking the variably spliced exon region (CD44s knock-in mice), and in knock-in strains with specific isoform expression (CD44v3-10 and CD44v4-10), as well as in double knock-in mice, expressing CD44v3-10 and CD44s. RESULTS: Expression of total CD44 and CD44v3 mRNA following femoral artery ligation was increased, accompanied by increased mRNA levels of the CD44-relevant splicing factors Tra2-beta1 and SRm160. CD44v3-expression was limited to the vessel wall of growing collateral arteries. Perfusion restoration was significantly reduced in mice lacking the variably spliced exon region (CD44s):20.1 ± 1.3%, compared to the background strain: 57.3 ± 2.2%. Mice expressing exon v3 (CD44v3-10) showed perfusion percentages of 25.9 ± 1.1%, compared to mice lacking this exon (CD44v4-10):19.1 ± 0.7%. Combined expression of CD44v3 and CD44s further improved perfusion restoration: 33.1 ± 2.6%. CONCLUSION: Total CD44 and CD44v3 mRNA are upregulated during arteriogenesis. The absence of the variably spliced exon region impairs arteriogenesis. Presence of exon v3 of CD44 results in improved arteriogenesis. Expression of CD44s and CD44v3 provides a synergistic effect on arteriogenesis. As this combined expression still resulted in hampered arteriogenesis, a specific role of exon v2 in arteriogenesis appears likely.

Forkhead box protein P1 as a downstream target of transforming growth factor-ß induces collagen synthesis and correlates with a more stable plaque phenotype.

OBJECTIVE: Atherosclerosis is an inflammatory disease, modulated by plaque stabilizing and de-stabilizing cell populations such as infiltrating monocytes and vascular smooth muscle cells (vSMCs). Transcription factors regulating proliferation and differentiation of atherosclerosis relevant cell types are of interest in this context. The forkhead box transcription factor FoxP1 modulates monocyte differentiation. We studied FoxP1 expression in atherosclerotic tissue, correlated FoxP1 expression with plaque characteristics and identified associations between FoxP1 and plaque proteins. METHODS: 116 Atherosclerotic plaques from carotid endarterectomy samples were histologically classified (fibrous, fibroatheromatous, atheromatous) and subjected to semi-quantitative protein analysis. Macrophage, SMC content and intraplaque thrombus amount were determined histologically. FoxP1 expression was investigated by western blotting and immunohistochemistry. In addition FoxP1 was overexpressed in vitro to identify causal relations between FoxP1 and plaque proteins. RESULTS: FoxP1 expression was observed in SMCs, macrophages, endothelial cells and T-cells within the plaque. High SMC and collagen content correlated with increased FoxP1 isoform (72 kD and 95 kD) levels. 72 kD FoxP1 expression was lower in plaques containing intraplaque thrombus. FoxP1 correlated with active intraplaque TGFß signaling. In vitro stimulation of SMCs with TGFß resulted in increased FoxP1 levels. 72 kD FoxP1 correlated with expression of pro-fibrotic EGR-1 and increased Col1A1 expression. CONCLUSION: FoxP1 is expressed by different cell types in atherosclerotic lesions and associated with more stable plaque characteristics and intraplaque TGFß signaling. FoxP1 expression in vitro is induced by TGFß, resulting in increased collagen and EGR-1 expression, providing a mechanism for the observed association with a more stable plaque phenotype.

Arteriogenesis requires toll-like receptor 2 and 4 expression in bone-marrow derived cells.

Adaptive collateral growth (arteriogenesis) is an important protective mechanism against ischemic injury in patients with cardiovascular disease. Arteriogenesis involves enlargement of pre-existent arterial anastomoses and shares many mechanistic similarities with inflammatory processes. Although infusion of the Toll-like receptor (TLR) 4 ligand lipopolysaccharide (LPS) has shown to result in a significant stimulation of arteriogenesis and both Toll-like receptor 2 and 4 are involved in structural arterial adaptations, the requirement for TLRs in arteriogenesis has not yet been established. We therefore subjected TLR 2 null and TLR 4 defective mice to unilateral femoral artery occlusion. At 7 days, both TLR 2 null and TLR 4 defective mice showed a significant reduction (~35%) of collateral perfusion. Histological staining showed that TLR 2 and TLR 4 expression during arteriogenesis is mostly restricted to infiltrating leukocytes. To distinguish between the functional importance of vascular and leukocytic TLRs in arteriogenesis, cross-over bone marrow transplantation was performed 6 weeks before femoral artery occlusion. Perfusion measurements showed that transplantation of wild-type bone marrow into TLR 2 null and TLR 4 defective mice rescued the impaired arteriogenesis, while injection of TLR 2 null and TLR 4 defective bone marrow into wild-type mice significantly reduced collateral vessel growth to levels of TLR null/defective mice. RT-PCR analysis demonstrated a significant upregulation of two endogenous TLR ligands EDA and Hsp60 (91.7 fold and 1.9 fold respectively) in regions of collateral vessel formation. This study illustrates the involvement of TLR 2 and TLR 4 in adaptive collateral artery growth and shows the importance of TLR 2 and 4 expression by bone-marrow derived cells for this process.

Blocking interferon {beta} stimulates vascular smooth muscle cell proliferation and arteriogenesis.

Increased interferon (IFN)-ß signaling in patients with insufficient coronary collateralization and an inhibitory effect of IFNß on collateral artery growth in mice have been reported. The mechanisms of IFNß-induced inhibition of arteriogenesis are unknown. In stimulated monocytes from patients with chronic total coronary artery occlusion and decreased arteriogenic response, whole genome expression analysis showed increased expression of IFNß-regulated genes. Immunohistochemically, the IFNß receptor was localized in the vascular media of murine collateral arteries. Treatment of vascular smooth muscle cells (VSMC) with IFNß resulted in an attenuated proliferation, cell-cycle arrest, and increased expression of cyclin-dependent kinase inhibitor-1A (p21). The growth inhibitory effect of IFNß was attenuated by inhibition of p21 by RNA interference. IFNß-treated THP1 monocytes showed enhanced apoptosis. Subsequently, we tested if collateral artery growth can be stimulated by inhibition of IFNß-signaling. RNA interference of the IFNß receptor-1 (IFNAR1) increased VSMC proliferation, cell cycle progression, and reduced p21 gene expression. IFNß signaling and FAS and TRAIL expression were attenuated in monocytes from IFNAR1(-/-) mice, indicating reduced monocyte apoptosis. Hindlimb perfusion restoration 1 week after femoral artery ligation was improved in IFNAR1(-/-) mice compared with wild-type mice as assessed by infusion of fluorescent microspheres. These results demonstrate that IFNß inhibits collateral artery growth and VSMC proliferation through p21-dependent cell cycle arrest and induction of monocyte apoptosis. Inhibition of IFNß stimulates VSMC proliferation and collateral artery growth.

Hyaluronic acid metabolism is increased in unstable plaques.

BACKGROUND: Hyaluronic acid is expressed in atherosclerotic lesions, but its exact role in atherosclerotic disease remains unknown. As degradation of hyaluronic acid by hyaluronidase into low molecular weight hyaluronic acid (LMW-HA) is associated with inflammation and Matrix Metalloproteinase (MMP)-9 activity, we hypothesized that hyaluronic acid metabolism is increased in plaques with unstable characteristics like large lipid core, high number of macrophages, MMP-9 activity, low collagen and smooth muscle cell content. MATERIALS AND METHODS: Protein was isolated from 68 carotid artery specimens. The adjacent plaque segment was characterized for the histological parameters: lipid core, macrophage, collagen, smooth muscle cell (SMC) content and the amount of intra-plaque thrombus. Hyaluronidase activity, total hyaluronic acid and LMW-HA expression, the standard hayaluronic acid receptor CD44s and the VEGF-A binding isoform CD44v3, MMP-9 activity and the plaque instability associated growth factor Vascular Endothial Growth Factor (VEGF)-A were analysed and correlated with histological characteristics. RESULTS: Hyaluronidase activity, LMW-HA and CD44 expression (CD44s, CD44v3) levels were increased in atheromatous plaques compared with fibrous plaques. Total hyaluronic acid did not correlate with plaque instability. MMP-9 activity correlated with CD44s, hyaluronidase and LMW-HA expression. CD44v3 correlated with the angiogenic factor VEGF-A. In vitro stimulation of macrophages with LMW-HA increased MMP-9 activity. CONCLUSIONS: We show for the first time that increased hyaluronic acid metabolism and elevated CD44 levels are associated with plaque destabilization potentially by increased MMP-9 activity and stimulation of angiogenesis.

Increased expression of the transforming growth factor-beta signaling pathway, endoglin, and early growth response-1 in stable plaques.

BACKGROUND AND PURPOSE: Unstable atherosclerotic plaques are characterized by increased macrophages and reduced smooth muscle cells (SMCs) and collagen content. Endoglin, an accessory transforming growth factor-beta (TGFbeta) receptor, is a modulator of TGFbeta signaling recently found to be expressed on SMCs in atherosclerotic plaques. Its function in plaque SMCs and plaque development is unknown. Early growth response-1 (EGR-1), a transcription factor downstream of TGFbeta, stimulates SMC proliferation and collagen synthesis. In atherosclerotic lesions, it is mainly expressed by SMCs. Therefore, we studied the TGFbeta, endoglin, and EGR-1 pathway in advanced atherosclerotic plaques in relation to plaque phenotype. METHODS: Human carotid atherosclerotic plaques (n=103) were collected from patients undergoing carotid endarterectomy. Histologically, plaques were analyzed for plaque characteristics, ie, collagen, macrophage and SMC content, and intraplaque thrombus. Intraplaque endoglin, pSmad (indicative for TGFbeta signaling), EGR-1, and TGFbeta levels were analyzed using Western blots and enzyme-linked immunosorbent assays, respectively. RESULTS: Higher endoglin and EGR-1 protein levels correlated positively with increased plaque collagen levels, increased smooth muscle cell content, and decreased intraplaque thrombi as well as TGFbeta signaling (pSmad). Although EGR-1 overexpression in vitro stimulated collagen synthesis, inhibiting endoglin resulted in lower EGR-1 levels, decreased SMC proliferation, and decreased collagen content. CONCLUSIONS: TGFbeta in human atherosclerotic plaques is active and signals through the TGFbeta/Smad pathway. For the first time, we show a strong association between endoglin and EGR-1, increased collagen and SMCs expression, decreased levels of intraplaque thrombosis, and a stable plaque phenotype.

Hyaluronic acid: targeting immune modulatory components of the extracellular matrix in atherosclerosis.

PURPOSE OF REVIEW: Atherosclerosis is a chronic inflammatory disease of the vessel wall. Although it has become common knowledge that hyaluronic acid (HA), an important component of the extracellular matrix, is strongly involved in atherosclerotic disease development it has only recently become evident that HA, instead of being a static matrix polymer, is an active modulator of proliferation and inflammation of the atherosclerotic plaque. This review discusses the regulatory capacity of HA in atherosclerotic lesions and its effects on plaque stability. RECENT FINDINGS: The mechanisms by which HA might alter plaque stability are diverse. It regulates cellular migration and proliferation, lipid accumulation, and intraplaque angiogenesis. Smooth muscle cell migration is enhanced upon accumulation of HA, potentially stabilizing atherosclerotic plaques. On the other hand, HA is an important ligand for CD44, which stimulates inflammatory cell recruitment to lesions, leukocyte migration and cell proliferation in atherosclerotic plaques. Furthermore, HA forms complexes with low density lipoproteins, and uptake of these complexes by macrophages is increased compared to native LDL, indicating a more detrimental effect on atherosclerosis. The dynamic functional role of HA might be based on the functional difference between short and larger size fragments of this polymer, with either an inflammatory or an anti-inflammatory besides a pro-mitogenic and anti-mitogenic effect. Low molecular weight HA has been shown to be pro-angiogenic, whereas high molecular weight HA has an anti-angiogenic effect. The cause of these differential actions might be that HA synthesis is regulated by three different genes: HAS1, 2 and 3 leading to different size HA products. These genes are specifically expressed under certain conditions, e.g. HAS1 and HAS3 are selectively induced in inflammation, suggesting an important role of their products in this process. SUMMARY: Hyaluronic acid is an active regulatory component of atherosclerotic lesions. Further studies are warranted to gain more insight into the mechanisms which decide on the role of HA in atherosclerosis and plaque stability.

Interferon-beta signaling is enhanced in patients with insufficient coronary collateral artery development and inhibits arteriogenesis in mice.

Stimulation of collateral artery growth in patients has been hitherto unsuccessful, despite promising experimental approaches. Circulating monocytes are involved in the growth of collateral arteries, a process also referred to as arteriogenesis. Patients show a large heterogeneity in their natural arteriogenic response on arterial obstruction. We hypothesized that circulating cell transcriptomes would provide mechanistic insights and new therapeutic strategies to stimulate arteriogenesis. Collateral flow index was measured in 45 patients with single-vessel coronary artery disease, separating collateral responders (collateral flow index, >0.21) and nonresponders (collateral flow index, < or 1). Isolated monocytes were stimulated with lipopolysaccharide or taken into macrophage culture for 20 hours to mimic their phenotype during arteriogenesis. Genome-wide mRNA expression analysis revealed 244 differentially expressed genes (adjusted P, <0.05) in stimulated monocytes. Interferon (IFN)-beta and several IFN-related genes showed increased mRNA levels in 3 of 4 cellular phenotypes from nonresponders. Macrophage gene expression correlated with stimulated monocytes, whereas resting monocytes and progenitor cells did not display differential gene regulation. In vitro, IFN-beta dose-dependently inhibited smooth muscle cell proliferation. In a murine hindlimb model, perfusion measured 7 days after femoral artery ligation showed attenuated arteriogenesis in IFN-beta-treated mice compared with controls (treatment versus control: 31.5+/-1.2% versus 41.9+/-1.9% perfusion restoration, P<0.01). In conclusion, patients with differing arteriogenic response as measured with collateral flow index display differential transcriptomes of stimulated monocytes. Nonresponders show increased expression of IFN-beta and its downstream targets, and IFN-beta attenuates proliferation of smooth muscle cells in vitro and hampers arteriogenesis in mice. Inhibition of IFN-beta signaling may serve as a novel approach for the stimulation of collateral artery growth.