Catherine M. Shanahan

Coupling of the nucleus and cytoplasm: Role of the LINC complex

The nuclear envelope defines the barrier between the nucleus and cytoplasm and features inner and outer membranes separated by a perinuclear space (PNS). The inner nuclear membrane contains specific integral proteins that include Sun1 and Sun2. Although the outer nuclear membrane (ONM) is continuous with the endoplasmic reticulum, it is nevertheless enriched in several integral membrane proteins, including nesprin 2 Giant (nesp2G), an 800-kD protein featuring an NH2-terminal actin-binding domain. A recent study (Padmakumar, V.C., T. Libotte, W. Lu, H. Zaim, S. Abraham, A.A. Noegel, J. Gotzmann, R. Foisner, and I. Karakesisoglou. 2005. J. Cell Sci. 118:3419–3430) has shown that localization of nesp2G to the ONM is dependent upon an interaction with Sun1. In this study, we confirm and extend these results by demonstrating that both Sun1 and Sun2 contribute to nesp2G localization. Codepletion of both of these proteins in HeLa cells leads to the loss of ONM-associated nesp2G, as does overexpression of the Sun1 lumenal domain. Both treatments result in the expansion of the PNS. These data, together with those of Padmakumar et al. (2005), support a model in which Sun proteins tether nesprins in the ONM via interactions spanning the PNS. In this way, Sun proteins and nesprins form a complex that links the nucleoskeleton and cytoskeleton (the LINC complex).

Human Vascular Smooth Muscle Cells Undergo Vesicle-Mediated Calcification in Response to Changes in Extracellular Calcium and Phosphate Concentrations: A Potential Mechanism for Accelerated Vascular Calcification in ESRD

Patients with ESRD have a high circulating calcium (Ca) x phosphate (P) product and develop extensive vascular calcification that may contribute to their high cardiovascular morbidity. However, the cellular mechanisms underlying vascular calcification in this context are poorly understood. In an in vitro model, elevated Ca or P induced human vascular smooth muscle cell (VSMC) calcification independently and synergistically, a process that was potently inhibited by serum. Calcification was initiated by release from living VSMC of membrane-bound matrix vesicles (MV) and also by apoptotic bodies from dying cells. Vesicles released by VSMC after prolonged exposure to Ca and P contained preformed basic calcium phosphate and calcified extensively. However, vesicles released in the presence of serum did not contain basic calcium phosphate, co-purified with the mineralization inhibitor fetuin-A and calcified minimally. Importantly, MV released under normal physiologic conditions did not calcify, and VSMC were also able to inhibit the spontaneous precipitation of Ca and P in solution. The potent mineralization inhibitor matrix Gla protein was found to be present in MV, and pretreatment of VSMC with warfarin markedly enhanced vesicle calcification. These data suggest that in the context of raised Ca and P, vascular calcification is a modifiable, cell-mediated process regulated by vesicle release. These vesicles contain mineralization inhibitors derived from VSMC and serum, and perturbation of the production or function of these inhibitors would lead to accelerated vascular calcification.

High expression of genes for calcification-regulating proteins in human atherosclerotic plaques.

Calcification is common in atheromatous plaques and may contribute to plaque rupture and subsequent thrombosis. However, little is known about the mechanisms which regulate the calcification process. Using in situ hybridization and immunohistochemistry we show that two bone-associated proteins, osteopontin (OP) and matrix Gla protein (MGP), are highly expressed in human atheromatous plaques. High levels of OP mRNA and protein were found in association with necrotic lipid cores and areas of calcification. The predominant cell type in these areas was the macrophage-derived foam cell, although some smooth muscle cells could also be identified. MGP was expressed uniformly by smooth muscle cells in the normal media and at high levels in parts of the atheromatous intima. Highest levels of this matrix-associated protein were found in lipid-rich areas of the plaque. The pattern of expression of these two genes contrasted markedly with that of calponin and SM22 alpha, genes expressed predominantly by differentiated smooth muscle cells and whose expression was generally confined to the media of the vessel. The postulated function of OP and MGP as regulators of calcification in bone and the high levels and colocalization of both in atheromatous plaques suggest they have an important role in plaque pathogenesis and stability.

Medial Localization of Mineralization-Regulating Proteins in Association With Mönckeberg’s Sclerosis Evidence for Smooth Muscle Cell–Mediated Vascular Calcification

BACKGROUND Calcification of the media of peripheral arteries is referred to as Mönckebergs sclerosis (MS) and occurs commonly in aged and diabetic individuals. Its pathogenesis is unknown, but its presence predicts risk of cardiovascular events and leg amputation in diabetic patients. Several studies have documented expression of bone-associated genes in association with intimal atherosclerotic calcification, leading to the suggestion that vascular calcification may be a regulated process with similarities to developmental osteogenesis. Therefore, we examined gene expression in vessels with MS to determine whether there was evidence for a regulated calcification process in the vessel media. METHODS AND RESULTS In situ hybridization, immunohistochemistry, and semiquantitative reverse-transcription polymerase chain reaction were used to examine the expression of mineralization-regulating proteins in human peripheral arteries with and without MS. MS occurred in direct apposition to medial vascular smooth muscle cells (VSMCs) in the absence of macrophages or lipid. These VSMCs expressed the smooth muscle-specific gene SM22alpha and high levels of matrix Gla protein but little osteopontin mRNA. Compared with normal vessels, vessels with MS globally expressed lower levels of matrix Gla protein and osteonectin, whereas alkaline phosphatase, bone sialoprotein, bone Gla protein, and collagen II, all indicators of osteogenesis/chondrogenesis, were upregulated. Furthermore, VSMCs derived from MS lesions exhibited osteoblastic properties and mineralized in vitro. CONCLUSIONS These data indicate that medial calcification in MS lesions is an active process potentially orchestrated by phenotypically modified VSMCs.

Apoptosis Regulates Human Vascular Calcification In Vitro: Evidence for Initiation of Vascular Calcification by Apoptotic Bodies

The mechanisms involved in the initiation of vascular calcification are not known, but matrix vesicles, the nucleation sites for calcium crystal formation in bone, are likely candidates, because similar structures have been found in calcified arteries. The regulation of matrix vesicle production is poorly understood but is thought to be associated with apoptotic cell death. In the present study, we investigated the role of apoptosis in vascular calcification. We report that apoptosis occurs in a human vascular calcification model in which postconfluent vascular smooth muscle cell (VSMC) cultures form nodules spontaneously and calcify after ≈28 days. Apoptosis occurred before the onset of calcification in VSMC nodules and was detected by several methods, including nuclear morphology, the TUNEL technique, and external display of phosphatidyl serine. Inhibition of apoptosis with the caspase inhibitor ZVAD.fmk reduced calcification in nodules by ≈40%, as measured by the cresolphthalein method and alizarin red staining. In addition, when apoptosis was stimulated in nodular cultures with anti-Fas IgM, there was a 10-fold increase in calcification. Furthermore, incubation of VSMC-derived apoptotic bodies with 45Ca demonstrated that, like matrix vesicles, they can concentrate calcium. These observations provide evidence that apoptosis precedes VSMC calcification and that apoptotic bodies derived from VSMCs may act as nucleating structures for calcium crystal formation.

Osteo/Chondrocytic Transcription Factors and Their Target Genes Exhibit Distinct Patterns of Expression in Human Arterial Calcification

Objective—Mineralization-regulating proteins are found deposited at sites of vascular calcification. However, the relationship between the onset of calcification in vivo and the expression of genes encoding mineralization-regulating proteins is unknown. This study aimed to determine the temporal and spatial pattern of expression of key bone and cartilage proteins as atherosclerotic calcification progresses. Methods and Results—Using reverse transcription-polymerase chain reaction on a panel of noncalcified and calcified human arterial samples, two classes of proteins could be identified: (1) Matrix Gla protein, osteonectin, osteoprotegerin, and aggrecan were constitutively expressed by vascular smooth muscle cells (VSMCs) in the normal vessel media but downregulated in calcified arteries whereas (2) alkaline phosphatase, bone sialoprotein, osteocalcin, and collagen II were expressed predominantly in the calcified vessel together with Cbfa1, Msx2, and Sox9, transcription factors that regulate expression of these genes. In the calcified plaque in situ hybridization identified subsets of VSMCs expressing osteoblast and chondrocyte-like gene expression profiles whereas osteoclast-like macrophages were present around sites of calcification. Conclusions—These observations suggest a sequence of molecular events in vascular calcification beginning with the loss of expression by VSMCs, of constitutive inhibitory proteins, and ending with expression by VSMCs and macrophages of chondrocytic, osteoblastic, and osteoclastic-associated proteins that orchestrate the calcification process.

Arterial Calcification in Chronic Kidney Disease: Key Roles for Calcium and Phosphate

Vascular calcification contributes to the high risk of cardiovascular mortality in chronic kidney disease (CKD) patients. Dysregulation of calcium (Ca) and phosphate (P) metabolism is common in CKD patients and drives vascular calcification. In this article, we review the physiological regulatory mechanisms for Ca and P homeostasis and the basis for their dysregulation in CKD. In addition, we highlight recent findings indicating that elevated Ca and P have direct effects on vascular smooth muscle cells (VSMCs) that promote vascular calcification, including stimulation of osteogenic/chondrogenic differentiation, vesicle release, apoptosis, loss of inhibitors, and extracellular matrix degradation. These studies suggest a major role for elevated P in promoting osteogenic/chondrogenic differentiation of VSMC, whereas elevated Ca has a predominant role in promoting VSMC apoptosis and vesicle release. Furthermore, the effects of elevated Ca and P are synergistic, providing a major stimulus for vascular calcification in CKD. Unraveling the complex regulatory pathways that mediate the effects of both Ca and P on VSMCs will ultimately provide novel targets and therapies to limit the destructive effects of vascular calcification in CKD patients.

SUN1 Interacts with Nuclear Lamin A and Cytoplasmic Nesprins To Provide a Physical Connection between the Nuclear Lamina and the Cytoskeleton

ABSTRACT Nuclear migration and positioning within cells are critical for many developmental processes and are governed by the cytoskeletal network. Although mechanisms of nuclear-cytoskeletal attachment are unclear, growing evidence links a novel family of nuclear envelope (NE) proteins that share a conserved C-terminal SUN (Sad1/UNC-84 homology) domain. Analysis of Caenorhabditis elegans mutants has implicated UNC-84 in actin-mediated nuclear positioning by regulating NE anchoring of a giant actin-binding protein, ANC-1. Here, we report the identification of SUN1 as a lamin A-binding protein in a yeast two-hybrid screen. We demonstrate that SUN1 is an integral membrane protein located at the inner nuclear membrane. While the N-terminal domain of SUN1 is responsible for detergent-resistant association with the nuclear lamina and lamin A binding, lamin A/C expression is not required for SUN1 NE localization. Furthermore, SUN1 does not interact with type B lamins, suggesting that NE localization is ensured by binding to an additional nuclear component(s), most likely chromatin. Importantly, we find that the luminal C-terminal domain of SUN1 interacts with the mammalian ANC-1 homologs nesprins 1 and 2 via their conserved KASH domain. Our data provide evidence of a physical nuclear-cytoskeletal connection that is likely to be a key mechanism in nuclear-cytoplasmic communication and regulation of nuclear position.

Isolation of gene markers of differentiated and proliferating vascular smooth muscle cells.

To isolate specific markers of both differentiated and proliferating vascular smooth muscle cells (VSMCs), we used the technique of differential cDNA screening using RNA from cultured rat aortic VSMCs. The tissue specificity of expression of all of the cDNAs isolated was determined by Northern analysis. We isolated seven distinct cDNAs that were more strongly expressed in freshly dispersed, differentiated, aortic VSMCs compared with dedifferentiated late-passage cells. These were the cDNAs for tropoelastin, a matrix protein; alpha-smooth muscle (SM) actin, gamma-SM actin, calponin, and phospholamban, which are all proteins associated with the contractile function of differentiated VSMCs; SM22 alpha, a smooth muscle-specific protein of unknown function, and CHIP28, a putative membrane channel protein that is not highly expressed in other SM tissues and may therefore be a new VSMC marker. Two cDNAs that were expressed preferentially in late-passage dedifferentiated VSMCs were also isolated. These were the cDNAs for osteopontin and matrix Gla protein (MGP). Like CHIP28, MGP was strongly expressed in aortic VSMCs but not in other types of tissues containing SM cells, suggesting that both have specific functions in vascular tissue. Osteopontin and MGP have both previously been isolated from developing bone. Their expression in proliferating VSMCs suggests that they may be involved in regulating the calcification that commonly occurs in vascular lesions. The set of cDNAs obtained extends the range of DNA probes that are available for identifying VSMCs and characterizing their phenotype in vivo by in situ hybridization. Therefore, they should aid in the analysis of gene expression during the development of vessel lesions.

Dialysis Accelerates Medial Vascular Calcification in Part by Triggering Smooth Muscle Cell Apoptosis

Background— Vascular calcification is associated with increased morbidity and mortality in stage V chronic kidney disease, yet its early pathogenesis and initiating mechanisms in vivo remain poorly understood. To address this, we quantified the calcium (Ca) load in arteries from children (10 predialysis, 24 dialysis) and correlated it with clinical, biochemical, and vascular measures. Methods and Results— Vessel Ca load was significantly elevated in both predialysis and dialysis and was correlated with the patients’ mean serum Ca×phosphate product. However, only dialysis patients showed increased carotid intima-media thickness and increased aortic stiffness, and calcification on computed tomography was present in only the 2 patients with the highest Ca loads. Importantly, predialysis vessels appeared histologically intact, whereas dialysis vessels exhibited evidence of extensive vascular smooth muscle cell (VSMC) loss owing to apoptosis. Dialysis vessels also showed increased alkaline phosphatase activity and Runx2 and osterix expression, indicative of VSMC osteogenic transformation. Deposition of the vesicle membrane marker annexin VI and vesicle component mineralization inhibitors fetuin-A and matrix Gla-protein increased in dialysis vessels and preceded von Kossa positive overt calcification. Electron microscopy showed hydroxyapatite nanocrystals within vesicles released from damaged/dead VSMCs, indicative of their role in initiating calcification. Conclusions— Taken together, this study shows that Ca accumulation begins predialysis, but it is the induction of VSMC apoptosis in dialysis that is the key event in disabling VSMC defense mechanisms and leading to overt calcification, eventually with clinically detectable vascular damage. Thus the identification of factors that lead to VSMC death in dialysis will be of prime importance in preventing vascular calcification.