Activin-like kinase receptor 1 (ALK1) in atherosclerotic lesions and vascular mesenchymal cells

OBJECTIVE: Activin-like kinase receptor 1 (ALK1) is a transforming growth factor (TGF)-beta type I receptor expressed in vascular mesenchyme, yet its function in vascular mesenchymal cells (VMC) is unclear. We examined ALK1 expression in human coronary atherosclerotic lesions and bovine and human VMC undergoing cellular condensation in vitro. We also examined the effect of activated ALK1 on cell proliferation and smooth muscle cell (SMC) differentiation. METHODS AND RESULTS: Our results showed that ALK1 was expressed in human coronary atherosclerotic lesions as determined by immunohistochemistry. ALK1 was also expressed in cellular condensations of bovine and human VMC as determined by real-time PCR and immunocytochemistry. Bone morphogenetic protein (BMP)-2, which is known to increase condensation size, increased ALK1 expression when induced from a BMP-2 adenoviral vector. In turn, activated ALK1 induced expression of matrix GLA protein (MGP), a BMP-2 inhibitor known to limit condensation size. Activated ALK1 enhanced proliferation of VMC as determined by 3H-thymidine incorporation, whereas MGP decreased proliferation. Activated ALK1 also enhanced expression of SMC lineage markers and ALK5, another TGF-beta type I receptor, as determined by immunoblotting, real-time PCR and immunocytochemistry. Anti-TGF-beta antibodies abolished expression of SMC markers in the presence of constitutively active ALK1, suggesting that ALK1 activation alone is not sufficient to promote SMC differentiation. CONCLUSIONS: We conclude that there is a balance between the actions of BMP-2 and MGP in the initiation of vascular mesenchymal cell condensation and SMC differentiation, and that targeting ALK1, BMP2 and/or MGP may lead to novel concepts of atherosclerosis treatment.     

Cell differentiation in vascular calcification

Summary Ectopic tissue formation is commonly found in calcified atherosclerotic plaques. This suggests that cell differentiation plays an important role in vascular calcification, even though the origin of the cells involved is unclear. Calcifying vascular cells (CVCs), derived from bovine aortic media, have been used as an in vitro model for vascular calcification. CVCs have many characteristics in common with bone cells, but there are also differences suggesting mechanisms that may be applicable to the problem of osteoporosis in the setting of vascular calcification. Matrix GLA protein (MGP) deficient mice develop severe vascular calcification and die prematurely from heart failure and/or aortic rupture. The molecular mechanism of MGP is unknown. It has been hypothesized that MGP acts as a calcification inhibitor by binding calcium, preventing mineral deposition in extracelular fluids near the saturation point for calcium and phosphate. Alternatively, MGP expression may be an attempt to regulate cell differentiation in the vascular wall, possibly by acting as an inhibitor to a factor able to induce cartilage and bone such as bone morphogenetic proteins (BMPs).     

Vascular calcification: expression patterns of the osteoblast-specific gene core binding factor alpha-1 and the protective factor matrix gla protein in human atherogenesis.

OBJECTIVE: Increasing evidence suggests that vascular calcification is a regulated process. We studied the vascular expression pattern of a key factor in mineralization and a counteracting, protective factor. Based on the phenotype of null mice, Core binding factor alpha-1 (Cbfa-1) plays a pivotal role in bone formation, whereas Matrix Gla Protein (MGP) is a potent inhibitor of vascular calcification. METHODS: We investigated the expression of MGP and Cbfa-1 in cultured, human monocytic cells, endothelial cells and smooth muscle cells (SMC), as well as in normal and atherosclerotic vessel specimens. RESULTS: In cultured cells MGP is expressed in endothelial cells and SMC, whereas Cbfa-1 mRNA is predominantly present in macrophages and to a lesser extent in SMC. In the normal vessel wall MGP expression is high at the luminal side and declines toward the center of the media, whereas Cbfa-1 is absent. Moderate, diffuse calcification of the aorta media was observed only in those regions where MGP is low or absent. In atherosclerotic lesions MGP is expressed in endothelial cells and SMC that form fibrous caps, but is never present in macrophages. Cbfa-1 is synthesized in regions without MGP, it is associated with calcified areas and Cbfa-1 may be considered a marker for osteoprogenitor-like cells in the vessel wall. CONCLUSIONS: Our observations on MGP expression confirm and extend published data and are consistent with a protective function of MGP. Cbfa-1 expression is absent in normal medial SMC and co-localizes with neointimal macrophages and focal calcifications.     

Proline and gamma-carboxylated glutamate residues in matrix Gla protein are critical for binding of bone morphogenetic protein-4

Arterial calcification is ubiquitous in vascular disease and is, in part, prevented by matrix Gla protein (MGP). MGP binds calcium ions through gamma-carboxylated glutamates (Gla residues) and inhibits bone morphogenetic protein (BMP)-2/-4. We hypothesized that a conserved proline (Pro)64 is essential for BMP inhibition. We further hypothesized that calcium binding by the Gla residues is a prerequisite for BMP inhibition. Site-directed mutagenesis was used to modify Pro64 and the Gla residues, and the effect on BMP-4 activity, and binding of BMP-4 and calcium was tested using luciferase reporter gene assays, coimmunoprecipitation, crosslinking, and calcium quantification. The results showed that Pro64 was critical for binding and inhibition of BMP-4 but not for calcium binding. The Gla residues were also required for BMP-4 binding but flexibility existed. As long as 1 Gla residue remained on each side of Pro64, the ability to bind and inhibit BMP-4 was preserved. Chelation of calcium ions by EDTA or warfarin treatment of cells led to loss of ability of MGP to bind BMP-4. Our results also showed that phenylalanine could replace Pro64 without loss of function and that zebrafish MGP, which lacks upstream Gla residues, did not function as a BMP inhibitor. The effect of MGP mutagenesis on vascular calcification was determined in calcifying vascular cells. Only MGP proteins with preserved ability to bind and inhibit BMP-4 prevented osteogenic differentiation and calcification. Together, our results suggest that BMP and calcium binding in MGP are independent but functionally intertwined processes and that the BMP binding is essential for prevention of vascular calcification.     

Pathophysiology of vascular calcification in chronic kidney disease

Patients with chronic kidney disease (CKD) on dialysis have 2- to 5-fold more coronary artery calcification than age-matched individuals with angiographically proven coronary artery disease. In addition to increased traditional risk factors, CKD patients also have a number of nontraditional cardiovascular risk factors that may play a prominent role in the pathogenesis of arterial calcification, including duration of dialysis and disorders of mineral metabolism. In histological specimens from the inferior epigastric artery of dialysis patients, we have found expression of the osteoblast differentiation factor core binding factor alpha-1 (Cbfa1) and several bone-associated proteins (osteopontin, bone sialoprotein, alkaline phosphatase, type I collagen) in both the intima and medial layers when calcification was present. In cultured vascular smooth muscle cells, the addition of pooled serum from dialysis patients (versus normal healthy controls) accelerated mineralization and increased expression of Cbfa1, osteopontin, and alkaline phosphatase to a similar magnitude as does beta-glycerophosphate alone. However, a lack of inhibitors of calcification may also be important. Dialysis patients with low levels of serum fetuin-A, a circulating inhibitor of mineralization, have increased coronary artery calcification and fetuin-A can inhibit mineralization of vascular smooth muscle cells in vitro. These data support that elevated levels of phosphorus and/or other potential uremic toxins may play an important role by transforming vascular smooth muscle cells into osteoblast-like cells, which can produce a matrix of bone collagen and noncollagenous proteins. This nidus can then mineralize if the balance of pro-mineralizing factors outweighs inhibitory factors.     

Monocytes initiate a cycle of leukocyte recruitment when cocultured with endothelial cells

During the development of atherosclerotic plaque, monocytes and T-lymphocytes are recruited to the arterial intima by endothelial cells (EC) lining the vessel. This process is associated with chronic arterial inflammation and requires the activation-dependent expression of adhesion receptors and chemokines on EC. Here we show that monocytes can activate cocultured EC so that they support the adhesion, activation and transmigration of a secondary bolus of flowing peripheral blood monocytes or lymphocytes. The number of adherent leukocytes and their behaviour was comparable to that seen on EC activated with tumour necrosis factor-alpha (TNF-alpha). Depending upon the duration of endothelial cell/monocyte coculture different patterns of adhesion receptors were utilised by leukocytes. After 4 h coculture, antibodies against E-selectin, P-selectin and vascular cell adhesion molecule-1 (VCAM-1) reduced mononuclear leukocyte adhesion. After 24 h coculture, antibodies against E-selectin and VCAM-1 but not P-selectin were effective. Immunofluorescence analysis confirmed that monocyte coculture induced endothelial expression of E-selectin and VCAM-1, while P-selectin was at the limit of detection. We conclude that EC stimulated by monocytes can support the adhesion of flowing mononuclear leukocytes. We hypothesise that this mode of EC activation and leukocyte recruitment could initiate a self-perpetuating cycle of inflammation that could be relevant to atherogenesis and other chronic inflammatory disease states.     

Regulation of RANKL-induced osteoclastic differentiation by vascular cells

Vascular calcification is a regulated process of biomineralization resembling osteogenesis. Many bone-related factors, including resorptive osteoclast-like cells, although in low abundance, have been found in calcified atherosclerotic lesions. The regulatory mechanisms governing them in the vasculature, however, are not clear. Previously, we found that calcifying vascular cells (CVC), a subpopulation of bovine aortic smooth muscle cells (BASMC), undergo osteoblastic differentiation and form mineralized nodules. Since osteoblasts and marrow stromal preosteoblasts regulate osteoclastic differentiation in bone, we hypothesized that vascular cells also regulate differentiation of osteoclastic precursors in the artery wall. Peripheral blood monocytes, which are osteoclast precursors, were co-cultured with CVC or BASMC. Results showed that monocytes co-cultured with both of the vascular cells yielded fewer resorption pits than monocytes cultured alone. Furthermore, monocytes co-cultured with CVC had fewer resorption pits than those co-cultured with BASMC. Conditioned media from the vascular cells also inhibited resorptive activity of monocytes suggesting that the inhibitory effect was mediated in part by soluble factors. Compared with BASMC, CVC had lower mRNA expression for osteopontin, which promotes osteoclast attachment, but greater mRNA expression for the soluble inhibitory cytokine, IL-18. Increased osteoclastic differentiation was observed when neutralizing antibody to IL-18 receptor was added to the cultures of preosteoclasts with CVC conditioned media. Osteoprotegerin, another osteoclast inhibitory cytokine, was expressed at similar levels in both cultures. These results suggest that vascular cells inhibit osteoclastic differentiation, and that CVC have greater inhibitory effects than BASMC.     

Crossveinless 2 regulates bone morphogenetic protein 9 in human and mouse vascular endothelium

The importance of morphogenetic proteins (BMPs) and their antagonists in vascular development is increasingly being recognized. BMP-4 is essential for angiogenesis and is antagonized by matrix Gla protein (MGP) and crossveinless 2 (CV2), both induced by the activin receptor like-kinase 1 (ALK1) when stimulated by BMP-9. In this study, however, we show that CV2 preferentially binds and inhibits BMP-9 thereby providing strong feedback inhibition for BMP-9/ALK1 signaling rather than for BMP-4/ALK2 signaling. CV2 disrupts complex formation involving ALK2, ALK1, BMP-4, and BMP-9 required for the induction of both BMP antagonists. It also limits VEGF expression, proliferation, and tube formation in ALK1-expressing endothelial cells. In vivo, CV2 deficiency translates into a dysregulation of vascular BMP signaling, resulting in an abnormal endothelium with increased endothelial cellularity and expression of lineage markers for mature endothelial cells. Thus, mutual regulation by BMP-9 and CV2 is essential in regulating the development of the vascular endothelium.     

Insulin-like growth factor-I regulates proliferation and osteoblastic differentiation of calcifying vascular cells via extracellular signal-regulated protein kinase and phosphatidylinositol 3-kinase pathways

Vascular calcification develops within atherosclerotic lesions and results from a process similar to osteogenesis. One of the paracrine regulators of bone-derived osteoblasts, insulin-like growth factor-I (IGF-I), is also present in atherosclerotic lesions. To evaluate its possible role in vascular calcification, we assessed its in vitro effects on proliferation and differentiation in calcifying vascular cells (CVCs), a subpopulation of bovine aortic medial cells. Results showed that IGF-I inhibited spontaneous CVC differentiation and mineralization as evidenced by decreased alkaline phosphatase (AP) activity and decreased matrix calcium incorporation, respectively. Furthermore, IGF-I inhibited the AP activity induced by bacterial lipopolysaccharide, TNF-alpha, or H2O2. It also induced CVC proliferation based on 3H-thymidine incorporation. Results from Northern analysis and tests using IGF-I analogs suggest that IGF-I effects are mediated through the IGF-I receptor. IGF-I also activated both the extracellular signal-regulated protein kinase (ERK) and phosphatidylinositol 3-kinase (PI3K) pathways. Inhibition of either the ERK or PI3K pathway reversed IGF-I effects on CVC proliferation and AP activity, suggesting a common downstream target. Overexpression of ERK activator also mimicked IGF-I inhibition of lipopolysaccharide-induced AP activity. These results suggest that IGF-I promotes proliferation and inhibits osteoblastic differentiation and mineralization of vascular cells via both ERK and PI3K pathways.     

BMP-2 gene expression and effects on human vascular smooth muscle cells

Bone morphogenetic proteins (BMPs) and their serine/threonine kinase receptors have been identified in atherosclerotic arteries and vascular smooth muscle cells, respectively. Thus, BMPs (the largest subfamily of the TGF-beta superfamily) have been implicated in the pathogenesis of atherosclerosis. However, the origins of BMP biosynthesis and the functional roles of BMP in blood vessels are unclear. The present study explored BMP-2 gene expression in various human blood vessels and vascular cell types. Functional in vitro studies were also performed to determine the effects of recombinant human BMP-2 on migration (transwell assay) and proliferation ([3H]-thymidine incorporation) of human aortic vascular smooth muscle cells (HASMC). RT-PCR experiments revealed BMP-2 gene expression in normal and atherosclerotic human arteries as well as cultured human aortic and coronary vascular smooth muscle cells, human umbilical vein endothelial cells (HUVECs) and human macrophages. In cellular migration studies, incubation with BMP-2 produced efficacious (相似文献   

Current concepts of vascular calcification

Vascular calcification, such as coronary and aortic calcification, is a significant feature of vascular pathology. Two distinct forms of vascular calcification are well recognized. One is medial calcification, which occurs between the cell layers of smooth muscle cells, and is related to aging, diabetes and chronic renal failure. The other is atherosclerotic calcification, which occurs in the intima during the development of atheromatous disease. It has been shown that statins inhibit the progression of calcification in the aortic valve and the coronary artery. We have found that statins inhibit calcification of human aortic smooth muscle cells, which is induced by incubating the cells in high-phosphate medium. We also found that this is mediated by inhibiting cellular apoptosis, an essential mechanism for calcification, not by inhibiting inorganic phosphate (Pi) uptake by sodium-dependent phosphate cotransporter (NPC). Besides apoptosis and Pi uptake, such proteins as osteoprotegerin (OPG), matrix Gla protein (MGP), Klotho, fetuin-A, and apoE have been shown to negatively affect vascular calcification. Many previous reports suggest that vascular calcification appears to be regulated by promoting factors, such as Pi, apoptosis, modified LDL, advanced glycation end products, oxidative stress, vitaminD3, glucocorticoid, cbfa-1, osteopontin, and inhibitory factors, such as OPG, MGP, Klotho, fetuin-A, PTH/PTHrP, pyrophosphate, statins, and bisphosphonates. The precise mechanism of vascular calcification is of interest.     

Apoptosis of vascular smooth muscle cells is induced by Fas ligand derived from endothelial cells.

Although Fas-mediated cell death may play a role in atherogenesis, causal data in support of this hypothesis are lacking. The present study investigated the possibility that endothelial cells are involved in vascular smooth muscle cell (VSMC) apoptosis via the Fas-FasL pathway, and hence in atherogenesis. FACS analysis detected FasL on the surface of human umbilical vein endothelial cells (HUVECs) and immunofluorescence staining of the HUVECs demonstrated high levels of FasL in the intracellular compartment. FasL was down-regulated 4 h after tumor necrosis factor (TNFalpha) treatment, coinciding with maximal surface expression of the adhesion molecules vascular cell adhesion molecule-1 and E-selectin. However, the down-regulation of FasL expression was transient, as surface expression returned within 24 h of TNFalpha treatment. When cocultured with VSMCs, the FasL-expressing EC could kill the VSMCs in a manner that could be blocked by recombinant Fas-Fc, deployed as a soluble receptor for Fas. Moreover, when human coronary arteries were studied with immunohistochemistry using G247-4 monoclonal antibody for the detection of FasL, few FasL positive EC were observed in diffuse intimal thickening. In contrast, endothelium overlying the plaque showed prominent and uniform expression of FasL. These findings suggest that the Fas/FasL pathway can be used by EC to induce VSMC apoptosis in the atherosclerotic lesion.     

Thyroid hormone targets matrix Gla protein gene associated with vascular smooth muscle calcification

Thyroid hormones have marked cardiovascular effects in vivo. However, their direct effects on vascular smooth muscle cells have been unclear. Because thyroid hormones play critical roles in bone remodeling, we hypothesized that they are also associated with vascular smooth muscle calcification, one of the pathological features of vascular sclerosis. To test this hypothesis, we examined the effects of 3',3,5-triiodo-L-thyronine (T3) on the expression of calcification-associated genes in rat aortic smooth muscle cells (RAOSMCs). Quantitative RT-PCRs revealed that a physiological concentration of T3 (15 pmol/L free T3) increased mRNA level of matrix Gla protein (MGP), which acts as a potent inhibitor of vascular calcification in vivo, by 3-fold in RAOSMCs, as well as in cultured human coronary artery smooth muscle cells. In RAOSMCs transiently transfected with a luciferase reporter gene driven by the MGP promoter, T3 significantly stimulated luciferase activity. In addition, RNA interference against thyroid hormone receptor-alpha gene diminished the effect of T3 on MGP expression. Aortic smooth muscle tissues from methimazole-induced hypothyroid rats (400 mg/L drinking water; 4 weeks) also showed a 68% decrease in the MGP mRNA level, as well as a 33% increase in calcium content compared with that from the control euthyroid animals, whereas hyperthyroidism (0.2 mg T3/kg IP; 10 days) upregulated MGP mRNA by 4.5-fold and reduced calcium content by 11%. Our findings suggest that a physiological concentration of thyroid hormone directly facilitates MGP gene expression in smooth muscle cells via thyroid hormone nuclear receptors, leading to prevention of vascular calcification in vivo.     

NOTCH1 regulates matrix gla protein and calcification gene networks in human valve endothelium

Valvular and vascular calcification are common causes of cardiovascular morbidity and mortality. Developing effective treatments requires understanding the molecular underpinnings of these processes. Shear stress is thought to play a role in inhibiting calcification. Furthermore, NOTCH1 regulates vascular and valvular endothelium, and human mutations in NOTCH1 can cause calcific aortic valve disease. Here, we determined the genome-wide impact of altering shear stress and NOTCH signaling on human aortic valve endothelium. mRNA-sequencing of primary human aortic valve endothelial cells (HAVECs) with or without knockdown of NOTCH1, in the presence or absence of shear stress, revealed NOTCH1-dependency of the atherosclerosis-related gene connexin 40 (GJA5), and numerous repressors of endochondral ossification. Among these, matrix gla protein (MGP) is highly expressed in aortic valve and vasculature, and inhibits soft tissue calcification by sequestering bone morphogenetic proteins (BMPs). Altering NOTCH1 levels affected MGP mRNA and protein in HAVECs. Furthermore, shear stress activated NOTCH signaling and MGP in a NOTCH1-dependent manner. NOTCH1 positively regulated endothelial MGP in vivo through specific binding motifs upstream of MGP. Our studies suggest that shear stress activates NOTCH1 in primary human aortic valve endothelial cells leading to downregulation of osteoblast-like gene networks that play a role in tissue calcification.