VMA21 deficiency causes an autophagic myopathy by compromising V-ATPase activity and lysosomal acidification.

X-linked myopathy with excessive autophagy (XMEA) is a childhood-onset disease characterized by progressive vacuolation and atrophy of skeletal muscle. We show that XMEA is caused by hypomorphic alleles of the VMA21 gene, that VMA21 is the diverged human ortholog of the yeast Vma21p protein, and that like Vma21p it is an essential assembly chaperone of the V-ATPase, the principal mammalian proton pump complex. Decreased VMA21 raises lysosomal pH, which reduces lysosomal degradative ability and blocks autophagy. This reduces cellular free amino acids, which upregulates the mTOR pathway and mTOR-dependent macroautophagy, resulting in proliferation of large and ineffective autolysosomes that engulf sections of cytoplasm, merge together, and vacuolate the cell. Our results uncover macroautophagic overcompensation leading to cell vacuolation and tissue atrophy as a mechanism of disease.

Mutations in the V-ATPase Assembly Factor VMA21 Cause a Congenital Disorder of Glycosylation With Autophagic Liver Disease.

BACKGROUND AND AIMS: Vacuolar H+-ATP complex (V-ATPase) is a multisubunit protein complex required for acidification of intracellular compartments. At least five different factors are known to be essential for its assembly in the endoplasmic reticulum (ER). Genetic defects in four of these V-ATPase assembly factors show overlapping clinical features, including steatotic liver disease and mild hypercholesterolemia. An exception is the assembly factor vacuolar ATPase assembly integral membrane protein (VMA21), whose X-linked mutations lead to autophagic myopathy. APPROACH AND RESULTS: Here, we report pathogenic variants in VMA21 in male patients with abnormal protein glycosylation that result in mild cholestasis, chronic elevation of aminotransferases, elevation of (low-density lipoprotein) cholesterol and steatosis in hepatocytes. We also show that the VMA21 variants lead to V-ATPase misassembly and dysfunction. As a consequence, lysosomal acidification and degradation of phagocytosed materials are impaired, causing lipid droplet (LD) accumulation in autolysosomes. Moreover, VMA21 deficiency triggers ER stress and sequestration of unesterified cholesterol in lysosomes, thereby activating the sterol response element-binding protein-mediated cholesterol synthesis pathways. CONCLUSIONS: Together, our data suggest that impaired lipophagy, ER stress, and increased cholesterol synthesis lead to LD accumulation and hepatic steatosis. V-ATPase assembly defects are thus a form of hereditary liver disease with implications for the pathogenesis of nonalcoholic fatty liver disease.

Clusterin/Apolipoprotein J immunoreactivity is associated with white matter damage in cerebral small vessel diseases.

AIM: Brain clusterin is known to be associated with the amyloid-ß deposits in Alzheimer's disease (AD). We assessed the distribution of clusterin immunoreactivity in cerebrovascular disorders, particularly focusing on white matter changes in small vessel diseases. METHODS: Post-mortem brain tissues from the frontal or temporal lobes of a total of 70 subjects with various disorders including cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), cerebral amyloid angiopathy (CAA) and AD were examined using immunohistochemistry and immunofluorescence. We further used immunogold electron microscopy to study clusterin immunoreactivity in extracellular deposits in CADASIL. RESULTS: Immunostaining with clusterin antibodies revealed strong localization in arterioles and capillaries, besides cortical neurones. We found that clusterin immunostaining was significantly increased in the frontal white matter of CADASIL and pontine autosomal dominant microangiopathy and leukoencephalopathy subjects. In addition, clusterin immunostaining correlated with white matter pathology severity scores. Immunostaining in axons ranged from fine punctate deposits in single axons to larger confluent areas with numerous swollen axon bulbs, similar to that observed with known axon damage markers such as non-phosphorylated neurofilament H and the amyloid precursor protein. Immunofluorescence and immunogold electron microscopy experiments showed that whereas clusterin immunoreactivity was closely associated with vascular amyloid-ß in CAA, it was lacking within the granular osmiophilic material immunolabelled by NOTCH3 extracelluar domain aggregates found in CADASIL. CONCLUSIONS: Our results suggest a wider role for clusterin associated with white matter damage in addition to its ability to chaperone proteins for clearance via the perivascular drainage pathways in several disease states.

X-linked myopathy with excessive autophagy: a failure of self-eating.

Autophagic vacuolar myopathies (AVMs) are a group of disorders united by shared histopathological features on muscle biopsy that include the aberrant accumulation of autophagic vacuoles. The classic conditions that compose the AVMs include Pompe Disease, Danon Disease and X-linked myopathy with excessive autophagy (XMEA). Other disorders, including acquired myopathies like chloroquine toxicity, also have features of an autophagic myopathy. This review is focused on XMEA, a myopathy with onset of slowly progressive proximal weakness and elevated serum creatine kinase (2× to 20× normal) typically in the first decade of life. However, both late-adult onset and severe, sometimes lethal, neonatal cases also occur. Skeletal muscle pathology is characterized by numerous cytoplasmic autophagic vacuoles, complex muscle fiber splitting with internalization of capillaries, and complement C5b-9 deposition within vacuoles and along the sarcolemma. The autophagic vacuoles have sarcolemmal features. Mutations in the VMA21 gene at Xq28 cause XMEA by reducing the activity of lysosomal hydrolases. The VMA21 protein regulates the assembly of the V-ATPase required to acidify the lysosome. Increased lysosomal pH and poor degradation of cellular debris may secondarily induce autophagy, the net effect being accumulation of autophagolysosomes. The relationship of XMEA to other lysosomal disorders of muscle and potential therapeutic interventions for XMEA are discussed.

Hypomorphic Notch 3 alleles link Notch signaling to ischemic cerebral small-vessel disease.

The most common monogenic cause of small-vessel disease leading to ischemic stroke and vascular dementia is the neurodegenerative syndrome cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), which is associated with mutations in the Notch 3 receptor. CADASIL pathology is characterized by vascular smooth muscle cell degeneration and accumulation of diagnostic granular osmiophilic material (GOM) in vessels. The functional nature of the Notch 3 mutations causing CADASIL and their mechanistic connection to small-vessel disease and GOM accumulation remain enigmatic. To gain insight into how Notch 3 function is linked to CADASIL pathophysiology, we studied two phenotypically distinct mutations, C455R and R1031C, respectively associated with early and late onset of stroke, by using hemodynamic analyses in transgenic mouse models, receptor activity assays in cell culture, and proteomic examination of postmortem human tissue. We demonstrate that the C455R and R1031C mutations define different hypomorphic activity states of Notch 3, a property linked to ischemic stroke susceptibility in mouse models we generated. Importantly, these mice develop osmiophilic deposits and other age-dependent phenotypes that parallel remarkably the human condition. Proteomic analysis of human brain vessels, carrying the same CADASIL mutations, identified clusterin and collagen 18 α1/endostatin as GOM components. Our findings link loss of Notch signaling with ischemic cerebral small-vessel disease, a prevalent human condition. We determine that CADASIL pathophysiology is associated with hypomorphic Notch 3 function in vascular smooth muscle cells and implicate the accumulation of clusterin and collagen 18 α1/endostatin in brain vessel pathology.

The gene disrupted in Marinesco-Sjögren syndrome encodes SIL1, an HSPA5 cochaperone.

We identified the gene underlying Marinesco-Sjögren syndrome, which is characterized by cerebellar ataxia, progressive myopathy and cataracts. We identified four disease-associated, predicted loss-of-function mutations in SIL1, which encodes a nucleotide exchange factor for the heat-shock protein 70 (HSP70) chaperone HSPA5. These data, together with the similar spatial and temporal patterns of tissue expression of Sil1 and Hspa5, suggest that disturbed SIL1-HSPA5 interaction and protein folding is the primary pathology in Marinesco-Sjögren syndrome.

Monoclonal antibodies selective for α-synuclein oligomers/protofibrils recognize brain pathology in Lewy body disorders and α-synuclein transgenic mice with the disease-causing A30P mutation.

Inclusions of intraneuronal alpha-synuclein (α-synuclein) can be detected in brains of patients with Parkinson's disease and dementia with Lewy bodies. The aggregation of α-synuclein is a central feature of the disease pathogenesis. Among the different α-synuclein species, large oligomers/protofibrils have particular neurotoxic properties and should therefore be suitable as both therapeutic and diagnostic targets. Two monoclonal antibodies, mAb38F and mAb38E2, with high affinity and strong selectivity for large α-synuclein oligomers were generated. These antibodies, which do not bind amyloid-beta or tau, recognize Lewy body pathology in brains from patients with Parkinson's disease and dementia with Lewy bodies and detect pathology earlier in α-synuclein transgenic mice than linear epitope antibodies. An oligomer-selective sandwich ELISA, based on mAb38F, was set up to analyze brain extracts of the transgenic mice. The overall levels of α-synuclein oligomers/protofibrils were found to increase with age in these mice, although the levels displayed a large interindividual variation. Upon subcellular fractionation, higher levels of α-synuclein oligomers/protofibrils could be detected in the endoplasmic reticulum around the age when behavioral disturbances develop. In summary, our novel oligomer-selective α-synuclein antibodies recognize relevant pathology and should be important tools to further explore the pathogenic mechanisms in Lewy body disorders. Moreover, they could be potential candidates both for immunotherapy and as reagents in an assay to assess a potential disease biomarker.

White matter degeneration with Unverricht-Lundborg progressive myoclonus epilepsy: a translational diffusion-tensor imaging study in patients and cystatin B-deficient mice.

PURPOSE: To study white matter (WM) changes in patients with Unverricht-Lundborg progressive myoclonus epilepsy (EPM1) caused by mutations in the cystatin B gene and in the cystatin B-deficient (Cstb-/-) mouse model and to validate imaging findings with histopathologic analysis of mice. MATERIALS AND METHODS: Informed consent was obtained and the study was approved by an institutional ethics committee. Animal work was approved by the Animal Experiment Board of Finland. Diffusion-tensor imaging and tract-based spatial statistics (TBSS) were used to compare fractional anisotropic (FA) results and axial, radial, and mean diffusion among patients with EPM1 (n = 19) and control subjects (n = 18). Ex vivo diffusion-tensor imaging and TBSS were used to compare Cstb-/- mice (n = 9) with wild controls (n = 4). Areas of FA decrease in mice were characterized by means of immunohistochemical analysis and transmission electron microscopy. Student t test statistics were applied to report significant findings (threshold-free cluster enhancement, P < .05). RESULTS: Patients with EPM1 showed significantly (P < .05) reduced FA and increased radial and mean diffusion in all major WM tracts compared with those of control subjects, shown as global FA decrease along the TBSS skeleton (0.41 ± 0.03 vs 0.45 ± 0.02, respectively; P < 5 × 10(-6)). Cstb-/- mice exhibited significantly reduced FA (P < .05) and antimyelin basic protein staining. Transmission electron microscopy revealed degenerating axons in Cstb-/- mice vs controls (979 axons counted, 51 degenerating axons; 2.09 ± 0.29 per field vs 1072 axons counted, nine degenerating axons; 0.48 ± 0.19 per field; P = .002). CONCLUSION: EPM1 is characterized by widespread alterations in subcortical WM, the thalamocortical system, and the cerebellum, which result in axonal degeneration and WM loss. These data suggest that motor disturbances and other symptoms in patients with EPM1 involve not only the cortical system but also the thalamocortical system and cerebellum.

VMA21 deficiency prevents vacuolar ATPase assembly and causes autophagic vacuolar myopathy.

X-linked Myopathy with Excessive Autophagy (XMEA) is a childhood onset disease characterized by progressive vacuolation and atrophy of skeletal muscle. We show that XMEA is caused by hypomorphic alleles of the VMA21 gene, that VMA21 is the diverged human ortholog of the yeast Vma21p protein, and that like Vma21p, VMA21 is an essential assembly chaperone of the vacuolar ATPase (V-ATPase), the principal mammalian proton pump complex. Decreased VMA21 raises lysosomal pH which reduces lysosomal degradative ability and blocks autophagy. This reduces cellular free amino acids which leads to downregulation of the mTORC1 pathway, and consequent increased macroautophagy resulting in proliferation of large and ineffective autolysosomes that engulf sections of cytoplasm, merge, and vacuolate the cell. Our results uncover a novel mechanism of disease, namely macroautophagic overcompensation leading to cell vacuolation and tissue atrophy.

Novel mutations consolidate KCTD7 as a progressive myoclonus epilepsy gene.

BACKGROUND: The progressive myoclonus epilepsies (PMEs) comprise a group of clinically and genetically heterogeneous disorders characterised by myoclonus, epilepsy, and neurological deterioration. This study aimed to identify the underlying gene(s) in childhood onset PME patients with unknown molecular genetic background. METHODS: Homozygosity mapping was applied on genome-wide single nucleotide polymorphism data of 18 Turkish patients. The potassium channel tetramerisation domain-containing 7 (KCTD7) gene, previously associated with PME in a single inbred family, was screened for mutations. The spatiotemporal expression of KCTD7 was assessed in cellular cultures and mouse brain tissue. RESULTS: Overlapping homozygosity in 8/18 patients defined a 1.5 Mb segment on 7q11.21 as the major candidate locus. Screening of the positional candidate gene KCTD7 revealed homozygous missense mutations in two of the eight cases. Screening of KCTD7 in a further 132 PME patients revealed four additional mutations (two missense, one in-frame deletion, and one frameshift-causing) in five families. Eight patients presented with myoclonus and epilepsy and one with ataxia, the mean age of onset being 19 months. Within 2 years after onset, progressive loss of mental and motor skills ensued leading to severe dementia and motor handicap. KCTD7 showed cytosolic localisation and predominant neuronal expression, with widespread expression throughout the brain. None of three polypeptides carrying patient missense mutations affected the subcellular distribution of KCTD7. DISCUSSION: These data confirm the causality of KCTD7 defects in PME, and imply that KCTD7 mutation screening should be considered in PME patients with onset around 2 years of age followed by rapid mental and motor deterioration.

Prolonged myalgia in Sindbis virus infection: case description and in vitro infection of myotubes and myoblasts.

BACKGROUND: Sindbis virus (SINV) is a mosquito-borne alphavirus found in Eurasia, Africa, and Oceania. Clinical SINV infection is characterized by febrile rash and arthritis and sometimes prolonged arthralgia and myalgia. The pathophysiological mechanisms of musculoskeletal and rheumatic disease caused by SINV are inadequately understood. METHODS: We studied the muscle pathology of SINV infection ex vivo by examining a unique muscle biopsy obtained from a patient with chronic myalgia and arthralgia 6 months after acute SINV infection and assessed potential genetic predisposing factors by determining the human leukocyte antigen (HLA) and complement factor C4 genes and proteins. In addition, we performed in vitro SINV infections of primary human myoblasts and myotubes. RESULTS: In the muscle biopsy we found evidence of muscle regeneration due to previous necrotic lesions likely caused by earlier SINV infection. We showed that human myoblasts and myotubes were susceptible in vitro for SINV infection as the cells became immunoreactive for viral antigens and cytopathic effect was observed. The patient was homozygous for HLA-B*35 alleles and heterozygous for HLA-DRB1*01 and HLA-DRB1*03 alleles and had total deficiency of C4B protein. CONCLUSIONS: This study provides new insights concerning pathological processes leading to chronic symptoms in SINV infection and demonstrates for the first time the susceptibility of human myogenic cells to SINV infection.

Identification of low molecular weight pyroglutamate A{beta} oligomers in Alzheimer disease: a novel tool for therapy and diagnosis.

N-terminally truncated Aß peptides starting with pyroglutamate (AßpE3) represent a major fraction of all Aß peptides in the brain of Alzheimer disease (AD) patients. AßpE3 has a higher aggregation propensity and stability and shows increased toxicity compared with full-length Aß. In the present work, we generated a novel monoclonal antibody (9D5) that selectively recognizes oligomeric assemblies of AßpE3 and studied the potential involvement of oligomeric AßpE3 in vivo using transgenic mouse models as well as human brains from sporadic and familial AD cases. 9D5 showed an unusual staining pattern with almost nondetectable plaques in sporadic AD patients and non-demented controls. Interestingly, in sporadic and familial AD cases prominent intraneuronal and blood vessel staining was observed. Using a novel sandwich ELISA significantly decreased levels of oligomers in plasma samples from patients with AD compared with healthy controls were identified. Moreover, passive immunization of 5XFAD mice with 9D5 significantly reduced overall Aß plaque load and AßpE3 levels, and normalized behavioral deficits. These data indicate that 9D5 is a therapeutically and diagnostically effective monoclonal antibody targeting low molecular weight AßpE3 oligomers.

Gelsolin co-occurs with Lewy bodies in vivo and accelerates α-synuclein aggregation in vitro.

Deposition of fibrillar α-synuclein as Lewy bodies is the neuropathological hallmark of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Apart from α-synuclein, these intraneuronal inclusions contain over 250 different proteins. The actin binding protein gelsolin, has previously been suggested to be part of the Lewy body, but its potential role in α-synuclein aggregation remains unknown. Here, we studied the association between gelsolin and α-synuclein in brain tissue from PD and DLB patients as well as in a cell model for α-synuclein aggregation. Moreover, the potential effect of gelsolin on α-synuclein fibrillization was also investigated. Our data demonstrate that gelsolin co-occured with α-synuclein in Lewy bodies from affected human brain as well as with Lewy body-like inclusions in α-synuclein over expressing cells. Furthermore, in the presence of calcium chloride, gelsolin was found to enhance the aggregation rate of α-synuclein in vitro. Moreover, no apparent structural differences could be observed between fibrils formed in the presence or absence of gelsolin. Further studies on gelsolin and other Lewy body associated proteins are warranted to learn more about their potential role in the α-synuclein aggregation process.

alpha-Synuclein pathology in the spinal cord autonomic nuclei associates with alpha-synuclein pathology in the brain: a population-based Vantaa 85+ study.

In most subjects with Parkinson's disease and dementia with Lewy bodies, alpha-synuclein (alphaS) immunoreactive pathology is found not only in the brain but also in the autonomic nuclei of the spinal cord. However, neither has the temporal course of alphaS pathology in the spinal cord in relation to the brain progression been established, nor has the extent of alphaS pathology in the spinal cord been analyzed in population-based studies. Using immunohistochemistry, the frequency and distribution of alphaS pathology were assessed semiquantitatively in the brains and spinal cord nuclei of 304 subjects who were aged at least 85 in the population-based Vantaa 85+ study. alphaS pathology was common in the spinal cord; 102 (34%) subjects had classic alphaS pathology in the thoracic and/or sacral autonomic nuclei. Moreover, 134 (44%) subjects showed grain- or dot-like immunoreactivity in neuropil (mini-aggregates) without classic Lewy neurites or Lewy bodies (LBs). The latter type of alphaS accumulation is associated with age, but also the classic alphaS pathology was found more often in the oldest compared to the youngest age group. The severity of alphaS pathology in the spinal cord autonomic nuclei is significantly associated with the extent and severity of alphaS pathology in the brain. Of the subjects, 60% with moderate to severe thoracic alphaS pathology and up to 89% with moderate to severe sacral alphaS pathology had diffuse neocortical type of LB pathology in the brain. alphaS pathology exclusively in the spinal cord was rare. Our study indicates that in general alphaS pathology in the spinal cord autonomic nuclei is associated with similar pathology in the brain.

Differences in aberrant expression and splicing of sarcomeric proteins in the myotonic dystrophies DM1 and DM2.

Aberrant transcription and mRNA processing of multiple genes due to RNA-mediated toxic gain-of-function has been suggested to cause the complex phenotype in myotonic dystrophies type 1 and 2 (DM1 and DM2). However, the molecular basis of muscle weakness and wasting and the different pattern of muscle involvement in DM1 and DM2 are not well understood. We have analyzed the mRNA expression of genes encoding muscle-specific proteins and transcription factors by microarray profiling and studied selected genes for abnormal splicing. A subset of the abnormally regulated genes was further analyzed at the protein level. TNNT3 and LDB3 showed abnormal splicing with significant differences in proportions between DM2 and DM1. The differential abnormal splicing patterns for TNNT3 and LDB3 appeared more pronounced in DM2 relative to DM1 and are among the first molecular differences reported between the two diseases. In addition to these specific differences, the majority of the analyzed genes showed an overall increased expression at the mRNA level. In particular, there was a more global abnormality of all different myosin isoforms in both DM1 and DM2 with increased transcript levels and a differential pattern of protein expression. Atrophic fibers in DM2 patients expressed only the fast myosin isoform, while in DM1 patients they co-expressed fast and slow isoforms. However, there was no increase of total myosin protein levels, suggesting that aberrant protein translation and/or turnover may also be involved.

Bedside diagnosis of rippling muscle disease in CAV3 p.A46T mutation carriers.

Thirty-nine members, ages 1 to 67 years, of a Swedish family with rippling muscle disease (RMD) were investigated to assess genotype-phenotype correlations. Clinical, neurophysiological, and muscle morphological examinations were performed. Genetic analysis was performed in 38 individuals. Twenty-three patients had percussion-induced muscle mounding (PIMM) and percussion-induced rapid contractions (PIRC). Rippling and hyperCKemia were not found in all patients. Weakness was minor or absent. The electromyogram showed absence of electrical activity in ripples and PIMM, and muscle biopsy specimens confirmed caveolin-3 deficiency and absence of caveolae. Genetic analysis revealed a CAV3 c.G136A transition resulting in a p.A46T missense mutation in affected family members. The phenotype in these 23 cases of RMD with this mutation appears to be homogenous, benign, and nonprogressive. The presence of PIMM and PIRC seems to be diagnostic at all ages, whereas the absence of hyperCKemia and rippling does not exclude the diagnosis.

Pyroglutamate Abeta pathology in APP/PS1KI mice, sporadic and familial Alzheimer's disease cases.

The presence of Abeta(pE3) (N-terminal truncated Abeta starting with pyroglutamate) in Alzheimer's disease (AD) has received considerable attention since the discovery that this peptide represents a dominant fraction of Abeta peptides in senile plaques of AD brains. This was later confirmed by other reports investigating AD and Down's syndrome postmortem brain tissue. Importantly, Abeta(pE3) has a higher aggregation propensity, and stability, and shows an increased toxicity compared to full-length Abeta. We have recently shown that intraneuronal accumulation of Abeta(pE3) peptides induces a severe neuron loss and an associated neurological phenotype in the TBA2 mouse model for AD. Given the increasing interest in Abeta(pE3), we have generated two novel monoclonal antibodies which were characterized as highly specific for Abeta(pE3) peptides and herein used to analyze plaque deposition in APP/PS1KI mice, an AD model with severe neuron loss and learning deficits. This was compared with the plaque pattern present in brain tissue from sporadic and familial AD cases. Abundant plaques positive for Abeta(pE3) were present in patients with sporadic AD and familial AD including those carrying mutations in APP (arctic and Swedish) and PS1. Interestingly, in APP/PS1KI mice we observed a continuous increase in Abeta(pE3) plaque load with increasing age, while the density for Abeta(1-x ) plaques declined with aging. We therefore assume that, in particular, the peptides starting with position 1 of Abeta are N-truncated as disease progresses, and that, Abeta(pE3) positive plaques are resistant to age-dependent degradation likely due to their high stability and propensity to aggregate.

Notch signaling regulates platelet-derived growth factor receptor-beta expression in vascular smooth muscle cells.

Notch signaling is critically important for proper architecture of the vascular system, and mutations in NOTCH3 are associated with CADASIL, a stroke and dementia syndrome with vascular smooth muscle cell (VSMC) dysfunction. In this report, we link Notch signaling to platelet-derived growth factor (PDGF) signaling, a key determinant of VSMC biology, and show that PDGF receptor (PDGFR)-beta is a novel immediate Notch target gene. PDGFR-beta expression was upregulated by Notch ligand induction or by activated forms of the Notch receptor. Moreover, upregulation of PDGFR-beta expression in response to Notch activation critically required the Notch signal integrator CSL. In primary VSMCs, PDGFR-beta expression was robustly upregulated by Notch signaling, leading to an augmented intracellular response to PDGF stimulation. In newborn Notch3-deficient mice, PDGFR-beta expression was strongly reduced in the VSMCs that later develop an aberrant morphology. In keeping with this, PDGFR-beta upregulation in response to Notch activation was reduced also in Notch3-deficient embryonic stem cells. Finally, in VSMCs from a CADASIL patient carrying a NOTCH3 missense mutation, upregulation of PDGFR-beta mRNA and protein in response to ligand-induced Notch activation was significantly reduced. In sum, these data reveal a hierarchy for 2 important signaling systems, Notch and PDGF, in the vasculature and provide insights into how dysregulated Notch signaling perturbs VSMC differentiation and function.