X-ray Micro-Computed Tomography: An Emerging Technology to Analyze Vascular Calcification in Animal Models.

Vascular calcification describes the formation of mineralized tissue within the blood vessel wall, and it is highly associated with increased cardiovascular morbidity and mortality in patients with chronic kidney disease, diabetes, and atherosclerosis. In this article, we briefly review different rodent models used to study vascular calcification in vivo, and critically assess the strengths and weaknesses of the current techniques used to analyze and quantify calcification in these models, namely 2-D histology and the o-cresolphthalein assay. In light of this, we examine X-ray micro-computed tomography (µCT) as an emerging complementary tool for the analysis of vascular calcification in animal models. We demonstrate that this non-destructive technique allows us to simultaneously quantify and localize calcification in an intact vessel in 3-D, and we consider recent advances in µCT sample preparation techniques. This review also discusses the potential to combine 3-D µCT analyses with subsequent 2-D histological, immunohistochemical, and proteomic approaches in correlative microscopy workflows to obtain rich, multifaceted information on calcification volume, calcification load, and signaling mechanisms from within the same arterial segment. In conclusion we briefly discuss the potential use of µCT to visualize and measure vascular calcification in vivo in real-time.

FTI-277 inhibits smooth muscle cell calcification by up-regulating PI3K/Akt signaling and inhibiting apoptosis.

BACKGROUND: Vascular calcification is associated with increased cardiovascular morbidity and mortality in patients with atherosclerosis, diabetes and chronic kidney disease. However, no viable treatments for this condition have been identified. This study aimed to determine whether farnesyl transferase inhibitors (FTIs) can reduce vascular calcification and the mechanism by which this reduction occurs. RESULTS: We demonstrate that FTI-277 significantly inhibits phosphate-induced mineral deposition by vascular smooth muscle cells (VSMC) in vitro, prevents VSMC osteogenic differentiation, and increases mRNA expression of matrix Gla protein (MGP), an inhibitor of mineralization. FTI-277 increases Akt signaling in VSMC in short-term serum-stimulation assays and in long-term mineralization assays. In contrast, manumycin A has no effect on Akt signaling or mineralization. Co-incubation of VSMC with FTI-277 and SH6 (an Akt inhibitor) significantly reduces the inhibitory effect of FTI-277 on mineralization, demonstrating that FTI-277 inhibits calcification by activating Akt signaling. Over-expression of the constitutively active p110 sub-unit of PI3K in VSMC using adenovirus activates Akt, inhibits mineralization, suppresses VSMC differentiation and significantly enhances MGP mRNA expression. FTI-277 also inhibits phosphate-induced activation of caspase 3 and apoptosis of VSMC, and these effects are negated by co-incubation with SH6. Finally, using an ex vivo model of vascular calcification, we demonstrate that FTI-277 inhibits high phosphate-induced mineralization in aortic rings derived from rats with end-stage renal failure. CONCLUSIONS: Together, these results demonstrate that FTI-277 inhibits VSMC mineral deposition by up-regulating PI3K/Akt signaling and preventing apoptosis, suggesting that targeting farnesylation, or Akt specifically, may have therapeutic potential for the prevention of vascular calcification.

Sphingosine 1-phosphate activation of ERM contributes to vascular calcification.

Vascular calcification is the deposition of mineral in the artery wall by vascular smooth muscle cells (VSMCs) in response to pathological stimuli. The process is similar to bone formation and is an independent risk factor for cardiovascular disease. Given that ceramide and sphingosine 1-phosphate (S1P) are involved in cardiovascular pathophysiology and biomineralization, their role in VSMC matrix mineralization was investigated. During phosphate-induced VSMC mineralization, endogenous S1P levels increased accompanied by increased sphingosine kinase (SK) activity and increased mRNA expression of SK1 and SK2. Consistent with this, mineralization was increased by exogenous S1P, but decreased by C2-ceramide. Mechanistically, exogenous S1P stimulated ezrin-radixin-moesin (ERM) phosphorylation in VSMCs and ERM phosphorylation was increased concomitantly with endogenous S1P during mineralization. Moreover, inhibition of acid sphingomyelinase and ceramidase with desipramine prevented increased S1P levels, ERM activation, and mineralization. Finally, pharmacological inhibition of ERM phosphorylation with NSC663894 decreased mineralization induced by phosphate and exogenous S1P. Although further studies will be needed to verify these findings in vivo, this study defines a novel role for the SK-S1P-ERM pathways in phosphate-induced VSMC matrix mineralization and shows that blocking these pathways with pharmacological inhibitors reduces mineralization. These results may inform new therapeutic approaches to inhibit or delay vascular calcification.

Regulation of vascular smooth muscle cell calcification by syndecan-4/FGF-2/PKCα signalling and cross-talk with TGFß.

AIMS: Vascular calcification is a major cause of morbidity and mortality. Fibroblast growth factor-2 (FGF-2) plays an instructive role in osteogenesis and bone development, but its role in vascular calcification was unknown. Therefore, we investigated the involvement of FGF-2 in vascular calcification and determined the mechanism by which it regulates this process. METHODS AND RESULTS: We demonstrate that FGF-2 expression is increased in vascular smooth muscle cells (VSMCs) induced to deposit a mineralized matrix by incubation with ß-glycerophosphate. FGF-2 is also localized to sites of calcification within human atherosclerotic plaques. The expression of syndecan-4, a heparan sulfate proteoglycan which regulates FGF-2 signalling, is also increased in mineralizing VSMCs and co-localizes with FGF-2 in human calcified atherosclerotic plaques. Exogenous FGF-2 inhibits VSMC mineralization, and this inhibition is reduced when syndecan-4 expression is knocked-down using siRNA. Biochemical inhibition of FGFR signalling using a pan FGFR inhibitor (BGJ398) or knocking-down syndecan-4 expression in VSMCs using siRNA increases VSMC mineralization. These increases are prevented by inhibiting transforming growth factor-ß (TGFß) signalling with SB431542, suggesting cross-talk between FGF-2 and TGFß signalling is crucial for the regulation of VSMC mineralization. Syndecan-4 can also regulate FGF-2 signalling directly via protein kinase Cα (PKCα) activation. Biochemical inhibition of PKCα activity using Gö6976, or siRNA-mediated suppression of PKCα expression increases VSMC mineralization; this increase is also prevented with SB431542. Finally, the ability of FGF-2 to inhibit VSMC mineralization is reduced when PKCα expression is knocked-down. CONCLUSION: This is the first demonstration that syndecan-4 promotes FGF-2 signalling, and in turn, suppresses VSMC mineralization by down-regulating TGFß signalling. Our discoveries that FGF-2 and syndecan-4 expression is increased in mineralizing VSMCs and that PKCα regulates FGF-2 and TGFß signalling in VSMCs suggests that the syndecan-4/FGF-2/TGFß signalling axis could represent a new therapeutic target for vascular calcification.

Comparative quantification of the surfaceome of human multipotent mesenchymal progenitor cells.

Mesenchymal progenitor cells have great therapeutic potential, yet incomplete characterization of their cell-surface interface limits their clinical exploitation. We have employed subcellular fractionation with quantitative discovery proteomics to define the cell-surface interface proteome of human bone marrow mesenchymal stromal/stem cells (MSCs) and human umbilical cord perivascular cells (HUCPVCs). We compared cell-surface-enriched fractions from MSCs and HUCPVCs (three donors each) with adult mesenchymal fibroblasts using eight-channel isobaric-tagging mass spectrometry, yielding relative quantification on >6,000 proteins with high confidence. This approach identified 186 upregulated mesenchymal progenitor biomarkers. Validation of 10 of these markers, including ROR2, EPHA2, and PLXNA2, confirmed upregulated expression in mesenchymal progenitor populations and distinct roles in progenitor cell proliferation, migration, and differentiation. Our approach has delivered a cell-surface proteome repository that now enables improved selection and characterization of human mesenchymal progenitor populations.

Axl tyrosine kinase protects against tubulo-interstitial apoptosis and progression of renal failure in a murine model of chronic kidney disease and hyperphosphataemia.

Chronic kidney disease (CKD) is defined as the progressive loss of renal function often involving glomerular, tubulo-interstitial and vascular pathology. CKD is associated with vascular calcification; the extent of which predicts morbidity and mortality. However, the molecular regulation of these events and the progression of chronic kidney disease are not fully elucidated. To investigate the function of Axl receptor tyrosine kinase in CKD we performed a sub-total nephrectomy and fed high phosphate (1%) diet to Axl+/+ and Axl-/- mice. Plasma Gas6 (Axl' ligand), renal Axl expression and downstream Akt signalling were all significantly up-regulated in Axl+/+ mice following renal mass reduction and high phosphate diet, compared to age-matched controls. Axl-/- mice had significantly enhanced uraemia, reduced bodyweight and significantly reduced survival following sub-total nephrectomy and high phosphate diet compared to Axl+/+ mice; only 45% of Axl-/- mice survived to 14 weeks post-surgery compared to 87% of Axl+/+ mice. Histological analysis of kidney remnants revealed no effect of loss of Axl on glomerular hypertrophy, calcification or renal sclerosis but identified significantly increased tubulo-interstitial apoptosis in Axl-/- mice. Vascular calcification was not induced in Axl+/+ or Axl-/- mice in the time frame we were able to examine. In conclusion, we identify the up-regulation of Gas6/Axl signalling as a protective mechanism which reduces tubulo-interstitial apoptosis and slows progression to end-stage renal failure in the murine nephrectomy and high phosphate diet model of CKD.

HGF/c-Met signalling promotes Notch3 activation and human vascular smooth muscle cell osteogenic differentiation in vitro.

OBJECTIVES: Vascular calcification is a major clinical problem and elucidating the underlying mechanism is important to improve the prognosis of patients with cardiovascular disease. We aimed to elucidate the role and mechanism of action of Hepatocyte Growth Factor (HGF)/c-Met signalling in vascular calcification and establish whether blocking this pathway could prevent mineralisation of vascular smooth muscle cells (VSMCs) in vitro. METHODS AND RESULTS: We demonstrate increased HGF secretion and c-Met up-regulation and phosphorylation during VSMC osteogenic differentiation. Adenoviral-mediated over-expression of HGF (AdHGF) in VSMCs accelerated mineralisation, shown by alizarin red staining, and significantly increased (45)Calcium incorporation (1.96 ± 0.54-fold [P < 0.05]) and alkaline phosphatase (ALP) activity (3.01 ± 0.8-fold [P < 0.05]) compared to controls. AdHGF also significantly elevated mRNA expression of bone-related proteins, Runx2, osteocalcin, BMP2 and osterix in VSMCs. AdHGF-accelerated mineralisation correlated with increased Akt phosphorylation, nuclear translocation of Notch3 intracellular domain (N3IC) and up-regulation of the Notch3 target protein, HES1. In contrast, adenoviral-mediated over-expression of the HGF antagonist, NK4, markedly attenuated VSMC mineralisation, and reduced c-Met phosphorylation, Akt activation and HES1 protein expression compared to AdHGF-treated cells. Furthermore, the Notch inhibitor, DAPT, attenuated N3IC nuclear translocation and AdHGF-induced mineralisation. CONCLUSION: We demonstrate HGF induces VSMC osteogenic differentiation via c-Met/Akt/Notch3 signalling, highlighting these pathways as potential targets for intervention of vascular calcification.

Apposite insulin-like growth factor (IGF) receptor glycosylation is critical to the maintenance of vascular smooth muscle phenotype in the presence of factors promoting osteogenic differentiation and mineralization.

Vascular calcification is strongly linked with increased morbidity and mortality from cardiovascular disease. Vascular calcification is an active cell-mediated process that involves the differentiation of vascular smooth muscle cells (VSMCs) to an osteoblast-like phenotype. Several inhibitors of this process have been identified, including insulin-like growth factor-I (IGF-I). In this study, we examined the role of the IGF receptor (IGFR) and the importance of IGFR glycosylation in the maintenance of the VSMC phenotype in the face of factors known to promote osteogenic conversion. IGF-I (25 ng/ml) significantly protected VSMCs from ß-glycerophosphate-induced osteogenic differentiation (p < 0.005) and mineral deposition (p < 0.01). Mevalonic acid depletion (induced by 100 nm cerivastatin) significantly inhibited these IGF protective effects (p < 0.01). Mevalonic acid depletion impaired IGFR processing, decreased the expression of mature IGFRs at the cell surface, and inhibited the downstream activation of Akt and MAPK. Inhibitors of N-linked glycosylation (tunicamycin, deoxymannojirimycin, and deoxynojirimycin) also markedly attenuated the inhibitory effect of IGF-I on ß-glycerophosphate-induced mineralization (p < 0.05) and activation of Akt and MAPK. These results demonstrate that alterations in the glycosylation of the IGFR disrupt the ability of IGF-I to protect against the osteogenic differentiation and mineralization of VSMCs by several interrelated mechanisms: decreased IGFR processing, reduced IGFR cell-surface expression, and reduced downstream signaling via the Akt and MAPK pathways. IGF-I thus occupies a critical position in the maintenance of normal VSMC phenotype and protection from factors known to stimulate vascular calcification.

Calcification is associated with loss of functional calcium-sensing receptor in vascular smooth muscle cells.

AIMS: Vascular calcification (VC) is highly correlated with increased morbidity and mortality in advanced chronic kidney disease (CKD) patients. Allosteric modulation of the calcium-sensing receptor (CaR) by calcimimetics inhibits VC in animal models of advanced CKD. Here, we investigated the expression of the CaR in the vasculature and tested the ability of calcimimetics to prevent vascular smooth muscle cell (VSMC) calcification in vitro. METHODS AND RESULTS: Immunohistochemical staining demonstrated that CaR protein is present in VSMC in normal, non-calcified human arteries. In contrast, low levels of CaR immunoreactivity were detected in atherosclerotic, calcified arteries. Immunfluorescence and immunoblotting revealed that CaR protein was also expressed by human and bovine VSMC in vitro. Acute stimulation of VSMC with increased Ca2+ stimulated extracellular signal-regulated kinase (ERK1/2) phosphorylation, suggesting that the VSMC CaR is functional. VSMC CaR expression decreased when these cells deposited a mineralized matrix or following 24 h incubation in mineralization medium with increased (i.e. 1.8 or 2.5 mM) Ca2+. Culturing VSMC in mineralization medium containing 1.8 and 2.5 mM Ca2+ or with the membrane-impermeant CaR agonist Gd3+ enhanced mineral deposition compared with that observed in 1.2 mM Ca2+. Over-expression of dominant-negative (R185Q) CaR enhanced, whereas the calcimimetic R-568 attenuated, VSMC mineral deposition. CONCLUSION: These results demonstrate that: (i) VSMCs express a functional CaR; (ii) a reduction in CaR expression is associated with increased mineralization in vivo and in vitro; (iii) calcimimetics decrease mineral deposition by VSMC. These data suggest that calcimimetics may inhibit the development of VC in CKD patients.

Cyclic stretch-induced TGFbeta1/Smad signaling inhibits adipogenesis in umbilical cord progenitor cells.

Human umbilical cord perivascular cells (HUCPVCs) can differentiate along numerous lineages making them a favourable cell source for tissue regeneration. However, how these cells respond to biomechanical forces is unclear. This study aimed to determine whether cyclic stretch could regulate adipogenic differentiation of HUCPVCs, and to elucidate the mechanism of this regulation. In adipogenic culture, HUCPVCs expressed the adipocyte-specific transcription factors PPARgamma and C/EBPalpha and accumulated cytoplasmic lipid droplets. Exposure of these cells to equibiaxial cyclic stretch (10%, 0.5 Hz) in the presence of adipogenic medium, increased Smad2 phosphorylation compared to static samples and inhibited the expression of adipocyte markers; ERK1/2 phosphorylation was not changed. Inhibiting TGFbeta1 signaling decreased Smad2 phosphorylation and prevented the inhibition of adipogenic differentiation by cyclic stretch. These results demonstrate that cyclic equibiaxial stretch regulates HUCPVC differentiation even in the presence of an adipogenic milieu and should be an important consideration in developing future progenitor cell therapies.

HtrA1 inhibits mineral deposition by osteoblasts: requirement for the protease and PDZ domains.

HtrA1 is a secreted multidomain protein with serine protease activity. In light of increasing evidence implicating this protein in the regulation of skeletal development and pathology, we investigated the role of HtrA1 in osteoblast mineralization and identified domains essential for this activity. We demonstrate increased HtrA1 expression in differentiating 2T3 osteoblasts prior to the appearance of mineralization. HtrA1 is subsequently down-regulated in fully mineralized cultures. The functional role of HtrA1 in matrix calcification was investigated using three complementary approaches. First, we transfected a full-length HtrA1 expression plasmid into 2T3 cells and showed that overexpression of HtrA1 delayed mineralization, reduced expression of Cbfa1 and collagen type I mRNA, and prevented BMP-2-induced mineralization. Second, knocking down HtrA1 expression using short interfering RNA induced mineral deposition by 2T3 cells. Third, by expressing a series of recombinant HtrA1 proteins, we demonstrated that the protease domain and the PDZ domain are essential for the inhibitory effect of HtrA1 on osteoblast mineralization. Finally, we tested whether HtrA1 cleaves specific matrix proteins that are known to regulate osteoblast differentiation, mineralization, and/or BMP-2 activity. Full-length recombinant HtrA1 cleaved recombinant decorin, fibronectin, and matrix Gla protein. Both the protease domain and the PDZ domain were necessary for the cleavage of matrix Gla protein, whereas the PDZ domain was not required for the cleavage of decorin or fibronectin. Type I collagen was not cleaved by recombinant HtrA1. These results suggest that HtrA1 may regulate matrix calcification via the inhibition of BMP-2 signaling, modulating osteoblast gene expression, and/or via the degradation of specific matrix proteins.

The role of endothelial cell attachment to elastic fibre molecules in the enhancement of monolayer formation and retention, and the inhibition of smooth muscle cell recruitment.

The endothelium is an essential modulator of vascular tone and thrombogenicity and a critical barrier between the vessel wall and blood components. In tissue-engineered small-diameter vascular constructs, endothelial cell detachment in flow can lead to thrombosis and graft failure. The subendothelial extracellular matrix provides stable endothelial cell anchorage through interactions with cell surface receptors, and influences the proliferation, migration, and survival of both endothelial cells and smooth muscle cells. We have tested the hypothesis that these desired physiological characteristics can be conferred by surface coatings of natural vascular matrix components, focusing on the elastic fiber molecules, fibrillin-1, fibulin-5 and tropoelastin. On fibrillin-1 or fibulin-5-coated surfaces, endothelial cells exhibited strong integrin-mediated attachment in static conditions (82% and 76% attachment, respectively) and flow conditions (67% and 78% cell retention on fibrillin-1 or fibulin-5, respectively, at 25 dynes/cm2), confluent monolayer formation, and stable functional characteristics. Adhesion to these two molecules also strongly inhibited smooth muscle cell migration to the endothelial monolayer. In contrast, on elastin, endothelial cells attached poorly, did not spread, and had markedly impaired functional properties. Thus, fibrillin-1 and fibulin-5, but not elastin, can be exploited to enhance endothelial stability, and to inhibit SMC migration within vascular graft scaffolds. These findings have important implications for the design of vascular graft scaffolds, the clinical performance of which may be enhanced by exploiting natural cell-matrix biology to regulate cell attachment and function.

Wnt/beta-catenin signaling stimulates chondrogenic and inhibits adipogenic differentiation of pericytes: potential relevance to vascular disease?

The aberrant differentiation of pericytes along the adipogenic, chondrogenic, and osteogenic lineages may contribute to the development and progression of several vascular diseases, including atherosclerosis and calcific vasculopathies. However, the mechanisms controlling pericyte differentiation and, in particular, adipogenic and chondrogenic differentiation are poorly defined. Wnt/beta-catenin signaling regulates cell differentiation during embryonic and postnatal development, and there is increasing evidence that it is involved in vascular pathology. Therefore, this study tested the hypothesis that Wnt/beta-catenin signaling regulates the chondrogenic and adipogenic differentiation of pericytes. We demonstrate that pericytes express several Wnt receptors, including LDL receptor-related proteins 5 and 6, and Frizzled 1 to 4 and 7, 8, and 10, and that Wnt/beta-catenin signaling is stimulated by both Wnt3a and LiCl. Furthermore, induction of Wnt/beta-catenin signaling by LiCl enhances chondrogenesis in pericyte pellet cultures in the presence of transforming growth factor-beta3, as demonstrated by increased Sox-9 expression and glycosaminoglycan accumulation into the matrix. In contrast, transduction of pericytes with a recombinant adenovirus encoding dominant-negative T-cell factor-4 (RAd/dnTCF), which blocks Wnt/beta-catenin signaling, inhibited chondrogenesis, leading to reduced Sox-9 and type II collagen expression and less glycosaminoglycan accumulation. Together, these data demonstrate that transforming growth factor-beta3 induces the chondrogenic differentiation of pericytes by inducing Wnt/beta-catenin signaling and T-cell factor-induced gene transcription. Induction of Wnt/beta-catenin signaling also attenuates adipogenic differentiation of pericytes in both pellet and monolayer cultures, as demonstrated by decreased staining with oil red O and reduced peroxisome proliferator-activated receptor gamma2 expression. This effect was negated by transduction of pericytes with RAd/dnTCF. Together, these results demonstrate that Wnt/beta-catenin signaling inhibits adipogenic and enhances chondrogenic differentiation of pericytes.

Axl/phosphatidylinositol 3-kinase signaling inhibits mineral deposition by vascular smooth muscle cells.

The calcification of blood vessels correlates with increased morbidity and mortality in patients with atherosclerosis, diabetes, and end-stage kidney disease. The receptor tyrosine kinase Axl is emerging as an important regulator of adult mammalian physiology and pathology. This study tests the hypothesis that Axl prevents the deposition of a calcified matrix by vascular smooth muscle cells (VSMCs) and that this occurs via the phosphatidylinositol 3-kinase (PI3K) signaling pathway. First, we demonstrate that Axl is expressed and phosphorylated in confluent VSMCs and that its expression is markedly downregulated as these cells calcify their matrix. Second, we demonstrate that overexpression of wild-type Axl, using recombinant adenoviruses, enhances Axl phosphorylation and downstream signaling via PI3K and Akt. Furthermore, overexpression of Axl significantly inhibits mineral deposition by VSMCs, as assessed by alizarin red staining and (45)Ca accumulation. Third, the addition of a PI3K inhibitor, wortmannin, negates the inhibition of mineralization by overexpression of wild-type Axl, suggesting that activation of downstream signaling via PI3K is crucial for its inhibitory activity. In contrast, Axl-mediated signaling is not enhanced by overexpression of kinase-dead Axl and mineralization is accelerated, although beta-glycerophosphate is still required for this effect. Finally, the caspase inhibitor zVAD.fmk attenuates the increased mineralization induced by kinase-dead Axl, suggesting that kinase-dead Axl stimulates mineralization by inhibiting the antiapoptotic effect of endogenous Axl. Together, these results demonstrate that signaling through Axl inhibits vascular calcification in vitro and suggest that therapeutics targeting this receptor may open up new avenues for the prevention of vascular calcification in vivo.

Alpha2(VIII) collagen substrata enhance endothelial cell retention under acute shear stress flow via an alpha2beta1 integrin-dependent mechanism: an in vitro and in vivo study.

BACKGROUND: Essential to tissue-engineered vascular grafts is the formation of a functional endothelial monolayer capable of resisting the forces of blood flow. This study targeted alpha2(VIII) collagen, a major component of the subendothelial matrix, and examined the ability of and mechanisms by which endothelial cells attach to this collagen under static and dynamic conditions both in vitro and in vivo. METHODS AND RESULTS: Attachment of human endothelial cells to recombinant alpha2(VIII) collagen was assessed in vitro under static and shear conditions of up to 100 dyne/cm2. Alpha2(VIII) collagen supported endothelial cell attachment in a dose-dependent manner, with an 18-fold higher affinity for endothelial cells compared with fibronectin. Cell attachment was significantly inhibited by function-blocking anti-alpha2 (56%) and -beta1 (98%) integrin antibodies but was not RGD dependent. Under flow, endothelial cells were retained at significantly higher levels on alpha2(VIII) collagen (53% and 51%) than either fibronectin (23% and 16%) or glass substrata (7% and 1%) at shear rates of 30 and 60 dyne/cm2, respectively. In vivo studies, using endothelialized polyurethane grafts, demonstrated significantly higher cell retention rates to alpha2(VIII) collagen-coated than to fibronectin-coated prostheses in the midgraft area (P < 0.05) after 24 hours' implantation in the caprine carotid artery. CONCLUSIONS: These studies demonstrate that alpha2(VIII) collagen has the potential to improve both initial cell attachment and retention of endothelial cells on vascular grafts in vivo, which opens new avenues of research into the development of single-stage endothelialized prostheses and the next generation of tissue-engineered vascular grafts.

Dexamethasone downregulates calcification-inhibitor molecules and accelerates osteogenic differentiation of vascular pericytes: implications for vascular calcification.

Vascular calcification is present in many pathological conditions and is recognized as a strong predictor of future cardiovascular events. Current evidence suggests that it is a regulated process involving inducing and inhibitory molecules. Glucocorticoids have great clinical importance as antiinflammatory drugs and can act as potent inducers of osteogenic differentiation in vitro. The effect of glucocorticoids on vascular cells in vivo remains obscure. Pericytes are pluripotent cells that can differentiate into osteoblasts, and recent evidence suggests that they could participate in vascular calcification. We hypothesized that the synthetic glucocorticoid dexamethasone would enhance the rate of pericyte differentiation and mineralization in vitro with a concomitant suppression of calcification-inhibitory molecules. Three weeks of dexamethasone treatment induced a 2-fold increase in (1) alkaline phosphatase activity, (2) calcium deposition, and (3) the number of nodules formed in vitro; and a reduction in the expression of matrix Gla protein (MGP), osteopontin (OPN), and vascular calcification-associated factor (VCAF) mRNAs. The glucocorticoid receptor antagonist Org 34116 abolished dexamethasone-accelerated pericyte differentiation, nodule formation, and mineralization. Data obtained using Org 34116, the transcription inhibitor actinomycin D, and the protein synthesis inhibitor cyclohexamide suggest that MGP, OPN, and VCAF mRNA abundance are controlled at different and multiple levels by dexamethasone. This is the first report showing that dexamethasone enhances the osteogenic differentiation of pericytes and downregulates genes associated with inhibition of mineralization. Our study highlights the need for further investigation into the long-term consequences of prolonged glucocorticoid therapy on vascular calcification.

Identification and characterization of vascular calcification-associated factor, a novel gene upregulated during vascular calcification in vitro and in vivo.

OBJECTIVE: Vascular calcification, with its increasing clinical sequelae, presents an important and unresolved dilemma in cardiac and vascular practice. We aimed to identify molecules involved in this process to develop strategies for treatment or prevention. METHODS AND RESULTS: Using subtractive hybridization, a novel cDNA, designated vascular calcification-associated factor (VCAF), has been isolated from a bovine retinal pericyte cDNA library generated during the differentiation and mineralization of these cells in vitro. RNA ligase-mediated rapid amplification of cDNA ends was used to compile the 740-bp bovine cDNA sequence. Database searching reveals that VCAF has novel nucleotide/amino acid sequences. RNA analysis confirms that VCAF is upregulated in mineralized pericytes and is present in human calcified arteries but not noncalcified arteries. Protein analysis using a VCAF antibody confirms the presence of an 18-kDa protein in calcified nodules but not in confluent pericytes. Adenoviral antisense VCAF gene delivery reduces VCAF protein levels and accelerates pericyte differentiation compared with controls. CONCLUSIONS: We demonstrate the isolation of a novel gene, VCAF, which is upregulated during vascular calcification in vitro and in vivo. Antisense VCAF gene delivery accelerates pericyte differentiation, implicating a role for VCAF in this clinically significant pathological process.

A novel hyaluronan-based biomaterial (Hyaff-11) as a scaffold for endothelial cells in tissue engineered vascular grafts.

Current prosthetic small diameter vascular grafts show poor long-term patency rates, leading to the pursuit of a biological alternative. Hyaff-11 is a hyaluronan-based biodegradable polymer developed for tissue-engineering applications. This study aimed to determine whether human vascular endothelial cells attach to Hyaff-11 scaffolds and produce a subendothelial matrix. Two forms of fibrous, non-woven Hyaff-11 scaffolds: unpressed and pressed felts, were analysed. Attachment of human venous endothelial cells was investigated after 1, 5, 10 and 20 days in culture using SEM and confocal microscopy. The deposition of subendothelial matrix components was investigated by immunofluorescent staining. We demonstrate that endothelial cells adhere to the individual fibres of both unpressed and pressed scaffolds: with a seeding density of 1 x 10(6) cells/cm(2), 94% of the cells attached to Hyaff-11 fibres after 24 h. The pressed material provided the best environment for cell growth, allowing the formation of a complete endothelial monolayer after 20 days. Furthermore, endothelial cells on Hyaff-11 pressed felts deposited an organised subendothelial matrix containing laminin, fibronectin, type IV and type VIII collagen. This work indicates Hyaff-11 based biopolymers as suitable scaffolds to promote endothelialisation within the next generation of vascular grafts.

Receptor tyrosine kinase Axl modulates the osteogenic differentiation of pericytes.

Vascular pericytes undergo osteogenic differentiation in vivo and in vitro and may, therefore, be involved in diseases involving ectopic calcification and osteogenesis. The purpose of this study was to identify factors that inhibit the entry of pericytes into this differentiation pathway. RNA was prepared from pericytes at confluence and after their osteogenic differentiation (mineralized nodules). Subtractive hybridization was conducted on polyA PCR-amplified RNA to isolate genes expressed by confluent pericytes that were downregulated in the mineralized nodules. The subtraction product was used to screen a pericyte cDNA library and one of the positive genes identified was Axl, the receptor tyrosine kinase. Northern and Western blotting confirmed that Axl was expressed by confluent cells and was downregulated in mineralized nodules. Western blot analysis demonstrated that confluent pericytes also secrete the Axl ligand, Gas6. Immunoprecipitation of confluent cell lysates with an anti-phosphotyrosine antibody followed by Western blotting using an anti-Axl antibody, demonstrated that Axl was active in confluent pericytes and that its activity could not be further enhanced by incubating the cells with recombinant Gas6. The addition of recombinant Axl-extracellular domain (ECD) to pericyte cultures inhibited the phosphorylation of Axl by endogenous Gas6 and enhanced the rate of nodule mineralization. These effects were inhibited by coincubation of pericytes with Axl-ECD and recombinant Gas6. Together these results demonstrate that activation of Axl inhibits the osteogenic differentiation of vascular pericytes.

Alternative splicing determines the domain structure of WWP1, a Nedd4 family protein.

Nedd-4-like proteins are E3 ubiquitin-ligase molecules which regulate key trafficking decisions, including targeting of proteins to proteosomes or lysosomes. Here we show that a human Nedd4 family gene, WWP1, is localized on 8q21 and generates at least six isoforms through alternative splicing. We show that alternative splicing affects the domain structure of WWP1, with forms that contain or lack an N-terminal C2 domain. Interestingly, the relative ratio of these forms varies in a tissue-specific manner. Other splice forms were also identified which may disrupt the structure of the C2 domain by removing its predicted C-terminal beta-strands. One splice form generates, through the introduction of a reading frame shift, a C2 domain-only form of WWP1. We discuss the hypothesis that regulation of splice site usage may modulate the activity of WWP1 and possibly other Nedd4 family proteins.