A reduction in the vascular smooth muscle cell focal adhesion component syndecan-4 is associated with abdominal aortic aneurysm formation.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a serious vascular disease for which there is no effective drug treatment. The incidence of AAA increases significantly as a subject ages, and the molecular mechanism of AAA formation remains elusive. In the present study, we investigated the role of syndecan-4 (SDC4), an important component of focal adhesions, in AAA formation and its association with phenotypic changes in vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: The protein expression levels of SDC4 were significantly decreased in human AAA tissue and those of an AAA mouse model. Moreover, SDC4 knockout (KO) in mice accelerated the formation and rupture of AAAs induced by angiotensin II (Ang II) and calcium chloride (CaCl2 ) Mechanistically, the decrease in SDC4 led to the transformation of cultured VSMCs from a contractile to a secretory phenotype. The RhoA-F/G-actin-myocardin-related transcription factor-A (MRTF-A) signalling pathway was shown to be involved in SDC4-dependent VSMC alteration. Sphingosine-1-phosphate (S1P), a G-protein-coupled receptor, attenuated the AAA formation in SDC4-KO and wild-type (WT) mice in response to Ang II and CaCl2 stimulation. CONCLUSION: We herein demonstrated that silencing SDC4 was associated with increased AAA formation and phenotypic changes in VSMCs via the RhoA-F/G-actin-MRTF-A pathway. These findings indicated that a reduction in SDC4 expression was an important pathological alteration and potential therapeutic target for AAA formation.

Lumican deficiency promotes pulmonary arterial remodeling.

Pulmonary arterial hypertension (PAH) is caused by progressive extracellular matrix disorganization and increased pulmonary vascular cell proliferation. Lumican is a member of the small leucine-rich proteoglycan family that controls cell proliferation, and is a potential endogenous modulator of TGF-ß signaling pathway. We show that the decreased lumican protein levels in pulmonary arterial smooth muscle cells (PASMCs) is related to the vascular remodeling and stiffening observed in PAH. The role of lumican in PASMC accumulation and activation in response to pulmonary vascular remodeling remains unclear and we hypothesized that the loss of lumican in PASMCs promotes the development of PAH. Our aim was to establish that lumican plays a pivotal role in modulating pathological vascular remodeling in humans using a rat model of monocrotaline-induced PAH and chronically hypoxic mice. We found that mice with a homozygous deletion of lumican (Lum-/-) showed severe pulmonary arterial remodeling and right ventricular hypertrophy in response to hypoxia, and these effects in mice with chronic hypoxia-induced pulmonary hypertension were successfully treated by the administration of a lumican C-terminal peptide (LumC13C-A, lumikine). We identified a mechanistic link by which lumican signaling prevents the activation of phosphorylated AKT, resulting in the suppression of PASMC proliferation. Lumican deficiency promotes pulmonary arterial remodeling. Administration of lumikine reverses the PAH pathogenesis caused by hypoxia-induced experimental PAH. Lumican is an antiproliferative target that functions to suppress pAKT activation during pathogenesis.

Morphometric analysis of thoracic aorta in Slc39a13/Zip13-KO mice.

Ehlers-Danlos syndrome (EDS) is a collection of inheritable diseases involving the musculoskeletal, integumentary and visual systems. Spondylodysplastic EDS-ZIP13 (spEDS-ZIP13: OMIM 612350) was recently defined as a new form of EDS. Although vasculitis has been found in many spEDS-ZIP13 patients, vascular pathology has not been included as a pathognomonic lesion of this type of EDS. We investigate the morphometry of the thoracic aorta in wild-type and Zip13-knockout (Zip13-KO) mice. Our assessment found abnormalities in the number and morphology of elastic and cellular components in the aortic wall, especially the tunica media, of Zip13-KO mice, indicating aortic fragility. Accordingly, our major findings (vascular smooth muscle cells with small nuclei, small percentage of elastic membrane area per tunica media, many large elastic flaps) should be considered vulnerable characteristics indicating fragility of the aorta in patients with spEDS-ZIP13.

Maternal food restriction modulates cerebrovascular structure and contractility in adult rat offspring: effects of metyrapone.

Although the effects of prenatal undernutrition on adult cardiovascular health have been well studied, its effects on the cerebrovascular structure and function remain unknown. We used a pair-fed rat model of 50% caloric restriction from day 11 of gestation to term, with ad libitum feeding after birth. We validated that maternal food restriction (MFR) stress is mediated by glucocorticoids by administering metyrapone, a corticosterone synthesis inhibitor, to MFR mothers at day 11 of gestation. At age 8 mo, offspring from Control, MFR, and MFR + Metyrapone groups were killed, and middle cerebral artery (MCA) segments were studied using vessel-bath myography and confocal microscopy. Colocalization of smooth muscle α-actin (SMαA) with nonmuscle (NM), SM1 and SM2 myosin heavy-chain (MHC) isoforms was used to assess smooth muscle phenotype. Our results indicate that artery stiffness and wall thickness were increased, pressure-evoked myogenic reactivity was depressed, and myofilament Ca(2+) sensitivity was decreased in offspring of MFR compared with Control rats. MCA from MFR offspring exhibited a significantly greater SMαA/NM colocalization, suggesting that the smooth muscle cells had been altered toward a noncontractile phenotype. MET significantly reversed the effects of MFR on stiffness but not myogenic reactivity, lowered SMαA/NM colocalization, and increased SMαA/SM2 colocalization. Together, our data suggest that MFR alters cerebrovascular contractility via both glucocorticoid-dependent and glucocorticoid-independent mechanisms.

Smad2 and myocardin-related transcription factor B cooperatively regulate vascular smooth muscle differentiation from neural crest cells.

RATIONALE: Vascular smooth muscle cell (VSMC) differentiation from neural crest cells (NCCs) is critical for cardiovascular development, but the mechanisms remain largely unknown. OBJECTIVE: Transforming growth factor-ß (TGF-ß) function in VSMC differentiation from NCCs is controversial. Therefore, we determined the role and mechanism of a TGF-ß downstream signaling intermediate Smad2 in NCC differentiation to VSMCs. METHODS AND RESULTS: By using Cre/loxP system, we generated a NCC tissue-specific Smad2 knockout mouse model and found that Smad2 deletion resulted in defective NCC differentiation to VSMCs in aortic arch arteries during embryonic development and caused vessel wall abnormality in adult carotid arteries where the VSMCs are derived from NCCs. The abnormalities included 1 layer of VSMCs missing in the media of the arteries with distorted and thinner elastic lamina, leading to a thinner vessel wall compared with wild-type vessel. Mechanistically, Smad2 interacted with myocardin-related transcription factor B (MRTFB) to regulate VSMC marker gene expression. Smad2 was required for TGF-ß-induced MRTFB nuclear translocation, whereas MRTFB enhanced Smad2 binding to VSMC marker promoter. Furthermore, we found that Smad2, but not Smad3, was a progenitor-specific transcription factor mediating TGF-ß-induced VSMC differentiation from NCCs. Smad2 also seemed to be involved in determining the physiological differences between NCC-derived and mesoderm-derived VSMCs. CONCLUSIONS: Smad2 is an important factor in regulating progenitor-specific VSMC development and physiological differences between NCC-derived and mesoderm-derived VSMCs.

Tbx1 genetically interacts with the transforming growth factor-ß/bone morphogenetic protein inhibitor Smad7 during great vessel remodeling.

RATIONALE: Growth and remodeling of the pharyngeal arch arteries are vital for the development of a mature great vessel system. Dysmorphogenesis of the fourth arch arteries can result in interruption of the aortic arch type B, typically found in DiGeorge syndrome. Tbx1 haploinsufficient embryos, which model DiGeorge syndrome, display fourth arch artery defects during formation of the vessels. Recovery from such defects is a documented yet unexplained phenotype in Tbx1 haploinsufficiency. OBJECTIVE: To understand the nature of fourth arch artery growth recovery in Tbx1 haploinsufficiency and its underlying genetic control. METHODS AND RESULTS: We categorized vessel phenotypes of Tbx1 heterozygotes as hypoplastic or aplastic at the conclusion of pharyngeal artery formation and compared these against the frequency of vessel defects scored at the end of great vessel development. The frequency of hypoplastic vessels decreased during embryogenesis, whereas no reduction of vessel aplasia was seen, implying recovery is attributable to remodeling of hypoplastic vessels. We showed that Smad7, an inhibitory Smad within the transforming growth factor-ß pathway, is regulated by Tbx1, is required for arch artery remodeling, and genetically interacts with Tbx1 in this process. Tbx1 and Tbx1;Smad7 haploinsufficiency affected several remodeling processes; however, concurrent haploinsufficiency particularly impacted on the earliest stage of vascular smooth muscle cell vessel coverage and subsequent fibronectin deposition. Conditional reconstitution of Smad7 with a Tbx1Cre driver indicated that the interaction between the 2 genes is cell autonomous. CONCLUSIONS: Tbx1 acts upstream of Smad7 controlling vascular smooth muscle and extracellular matrix investment of the fourth arch artery.

Decreased levels of embryonic retinoic acid synthesis accelerate recovery from arterial growth delay in a mouse model of DiGeorge syndrome.

RATIONALE: Loss of Tbx1 and decrease of retinoic acid (RA) synthesis result in DiGeorge/velocardiofacial syndrome (DGS/VCFS)-like phenotypes in mouse models, including defects in septation of the outflow tract of the heart and anomalies of pharyngeal arch-derived structures including arteries of the head and neck, laryngeal-tracheal cartilage, and thymus/parathyroid. Wild-type levels of T-box transcription factor (Tbx)1 and RA signaling are required for normal pharyngeal arch artery development. Recent studies have shown that reduction of RA or loss of Tbx1 alters the contribution of second heart field (SHF) progenitor cells to the elongating heart tube. OBJECTIVE: Here we tested whether Tbx1 and the RA signaling pathway interact during the deployment of the SHF and formation of the mature aortic arch. METHODS AND RESULTS: Molecular markers of the SHF, neural crest and smooth muscle cells, were analyzed in Raldh2;Tbx1 compound heterozygous mutants. Our results revealed that the SHF and outflow tract develop normally in Raldh2(+/-);Tbx1(+/-) embryos. However, we found that decreased levels of RA accelerate the recovery from arterial growth delay observed in Tbx1(+/-) mutant embryos. This compensation coincides with the differentiation of smooth muscle cells in the 4th pharyngeal arch arteries, and is associated with severity of neural crest cell migration defects observed in these mutants. CONCLUSIONS: Our data suggest that differences in levels of embryonic RA may contribute to the variability in great artery anomalies observed in DGS/VCFS patients.

An activating mutation in the PDGF receptor alpha results in embryonic lethality caused by malformation of the vascular system.

Platelet-derived growth factors (PDGF) and their receptors control cell proliferation, survival, and migration. To test the influence of an oncogenic mutation to embryonic development, a transgenic mouse line expressing PDGFRalpha (D842V) was established and analyzed. Most of the embryos die on embryonic day 12.5 due to massive hemorrhages in the trunk. In mesenchymal cells of mutant animals, proliferation is decreased while apoptosis is increased. Further analyses reveal that the aortic blood vessels are enlarged showing a reduced numbers of vascular smooth muscle cells (vSMC) around the aorta. We hypothesize that the process of aortic wall formation is impaired, leading to subsequent rupture and leakage of the blood vessel resulting in death of the embryos. We speculate that misexpression of PDGFRalpha in SMCs causes failure of vSMC recruitment to the aorta.

Absence of TGFbeta signaling in embryonic vascular smooth muscle leads to reduced lysyl oxidase expression, impaired elastogenesis, and aneurysm.

To address the requirement for TGFbeta signaling in the formation and maintenance of the vascular matrix, we employed lineage-specific mutation of the type II TGFbeta receptor gene (Tgfbr2) in vascular smooth muscle precursors in mice. In both neural crest- and mesoderm-derived smooth muscle, absence of TGFbeta receptor function resulted in a poorly organized vascular elastic matrix in late-stage embryos which was prone to dilation and aneurysm. This defect represents a failure to initiate formation of the elastic matrix, rather than a failure to maintain a preexisting matrix. In mutant tissue, lysyl oxidase expression was substantially reduced, which may contribute to the observed pathology.

Pulmonary vein, dorsal atrial wall and atrial septum abnormalities in podoplanin knockout mice with disturbed posterior heart field contribution.

The developing sinus venosus myocardium, derived from the posterior heart field, contributes to the atrial septum, the posterior atrial wall, the sino-atrial node, and myocardium lining the pulmonary and cardinal veins, all expressing podoplanin, a coelomic and myocardial marker. We compared development and differentiation of the myocardium and vascular wall of the pulmonary veins (PV), left atrial dorsal wall, and atrial septum in wild type with podoplanin knockout mouse embryos (E10.5-E18.5) by 3D reconstruction and immunohistochemistry. Expression of Nkx2.5 in the pulmonary venous myocardium changes from mosaic to positive during development pointing out a high proliferative rate compared with Nkx2.5 negative myocardium of the sino-atrial node and cardinal veins. In mutants, myocardium of the PVs, dorsal atrial wall and atrial septum was hypoplastic. The atrial septum and right-sided wall of the PV almost lacked interposed mesenchyme. Extension of smooth muscle cells into the left atrial body was diminished. We conclude that myocardium of the PVs, dorsal atrial wall, and atrial septum, as well as the smooth muscle cells, are derived from the posterior heart field regulated by podoplanin.

Knockout mice: is it just genetics? Effect of enriched housing on fibulin-4(+/-) mice.

BACKGROUND: Fibulin-4 is an extracellular matrix protein expressed by vascular smooth muscle cells that is essential for maintaining arterial integrity. Fibulin-4(-/-) mice die just before birth due to arterial hemorrhage, but fibulin-4(+/-) mice appear to be outwardly normal. Experiments were therefore performed to determine whether fibulin-4(+/-) mice display arterial pathologies on a microscopic scale. After preliminary experiments were performed, a second purpose developed, which was to test the hypothesis that any observed pathologies would be ameliorated by housing the animals in enriched cages. METHODOLOGY: Fibulin-4(+/-) and wild-type mice were housed either four/cage in standard cages or two per cage in larger cages, each cage containing a tunnel and a wheel. After three weeks the mice were sacrificed, and the aortas perfusion-fixed and excised for light and electron microscopy. PRINCIPLE FINDINGS: When the mice were in standard cages, localized regions of disorganized extracellular matrix and collagen fibers consistently appeared between some of the medial smooth muscle cells in the fibulin-4(+/-) mice. In the wild-type mice, the smooth muscle cells were closely connected to each other and the media was more compact. The number of disorganized regions per square mm was significantly greater for fibulin-4(+/-) mice (172+/-43 (SEM)) than for wild-type mice (15+/-8) (p<0.01, n = 8). When the mice were in enriched cages, the fibulin-4(+/-) mice showed significantly fewer disorganized regions than those in standard cages (35+/-12) (p<0.05, n = 8). The wild type mice also showed fewer disorganized regions (3+/-2), but this difference was not significant. CONCLUSIONS: These results indicate that arterial pathologies manifested in fibulin-4(+/-) mice can be reduced by enriching the housing conditions, and imply that appropriate environments may counteract the effects of some genetic deficiencies.

Pathologic aneurysmal dilation of the ascending aorta and dilation of the main pulmonary artery in patients with Kabuki syndrome: valve-sparing aortic root replacement.

We report the aneurysmal dilation of the ascending aorta and the main pulmonary artery in 2 children with Kabuki syndrome. In 1 patient, there was progressive aneurysmal dilation of the ascending aorta necessitating aortoplasty. Histologic examination of the resected aorta revealed disrupted and fragmented elastic fibers in the medial layer, along with mucinous degeneration of the aortic wall. This is the first recognition and report of these findings as part of the Kabuki syndrome.

Patterning of coronary arteries in wildtype and connexin43 knockout mice.

Abnormal patterning of coronary arteries (CAs) is a clinically significant problem, and as yet, few animal models have been systematically investigated for coronary patterning defects. Here we characterized coronary artery (CA) insertion and branching patterns of the proximal coronary stems in the hearts of wildtype and heterozygous connexin43 knockout (Cx43alpha1 KO) mice. This study entailed the use of high-resolution micro CT imaging for three-dimensional coronary reconstructions. MicroCT of 17 wildtype mice showed a remarkably consistent pattern of CA deployment in the normal mouse heart. Two main CA stems are inserted from the left and right into the aorta. The right coronary artery then branches immediately into the right main and the septal-conal branch, while the left coronary artery branches further distally into the circumflex and the anterior descending CA. This patterning of CA anatomy was confirmed by histology, and by using a vascular smooth muscle or endothelial cell specific lacZ reporter gene to delineate the CAs. A parallel analysis of 25 heterozygous Cx43alpha1 KO mouse hearts showed 22 had defects in patterning of the CAs. They exhibited a wide variation in CA anatomy, including abnormal origin and course of the main CA stems, multiple accessories, and dual septal-conal branches. Overall, these studies show loss of one Cx43alpha1 allele (haploinsufficiency) leads to a high incidence of coronary patterning defects. These findings suggest CA patterning is sensitive to Cx43alpha1 gene dosage.

The TGF beta activated kinase TAK1 regulates vascular development in vivo.

TGFbeta activated kinase 1 (TAK1) is a MAPKKK that in cell culture systems has been shown to act downstream of a variety of signaling molecules, including TGFbeta. Its role during vertebrate development, however, has not been examined by true loss-of-function studies. In this report, we describe the phenotype of mouse embryos in which the Tak1 gene has been inactivated by a genetrap insertion. Tak1 mutant embryos exhibit defects in the developing vasculature of the embryo proper and yolk sac. These defects include dilation and misbranching of vessels, as well as an absence of vascular smooth muscle. The phenotype of Tak1 mutant embryos is strikingly similar to that exhibited by loss-of-function mutations in the TGFbeta type I receptor Alk1 and the type III receptor endoglin, suggesting that TAK1 may be a major effector of TGFbeta signals during vascular development. Consistent with this view, we find that in zebrafish, morpholinos to TAK1 and ALK1 synergize to enhance the Alk1 vascular phenotype. Moreover, we show that overexpression of TAK1 is able to rescue the vascular defect produced by morpholino knockdown of ALK1. Taken together, these results suggest that TAK1 is probably an important downstream component of the TGFbeta signal transduction pathway that regulates vertebrate vascular development. In addition, as heterozygosity for mutations in endoglin and ALK1 lead to the human syndromes known as hereditary hemorrhagic telangiectasia 1 and 2, respectively, our results raise the possibility that mutations in human TAK1 might contribute to this disease.

Progressive vascular smooth muscle cell defects in a mouse model of Hutchinson-Gilford progeria syndrome.

Children with Hutchinson-Gilford progeria syndrome (HGPS) suffer from dramatic acceleration of some symptoms associated with normal aging, most notably cardiovascular disease that eventually leads to death from myocardial infarction and/or stroke usually in their second decade of life. For the vast majority of cases, a de novo point mutation in the lamin A (LMNA) gene is the cause of HGPS. This missense mutation creates a cryptic splice donor site that produces a mutant lamin A protein, termed "progerin," which carries a 50-aa deletion near its C terminus. We have created a mouse model for progeria by generating transgenics carrying a human bacterial artificial chromosome that harbors the common HGPS mutation. These mice develop progressive loss of vascular smooth muscle cells in the medial layer of large arteries, in a pattern very similar to that seen in children with HGPS. This mouse model should prove valuable for testing experimental therapies for this devastating disorder and for exploring cardiovascular disease in general.

Etiology of patent ductus arteriosus in dogs.

Patent ductus arteriosus (PDA) is the most common congenital heart disease in dogs and usually causes heart failure and death unless corrected at a young age. Previous histologic studies in a line of dogs derived from Miniature Poodles with hereditary PDA identified varying degrees of hypoplasia and asymmetry of ductus-specific smooth muscle and the presence of aortalike elastic tissue in the ductus wall sufficient to cause patency. To determine if similar structural abnormalities cause PDA in other dogs, serial-section, 3-dimensional histology of ductal architecture was studied in 8 non-Poodle purebred dogs with PDA with no immediate family history of PDA. Morphologic abnormalities were observed in 7 of 8 dogs with PDA and essentially were the same as those in dogs known to have a hereditary form of PDA. These findings suggest that apparently sporadic PDA in these breeds is caused by a genetic defect in the structure of the ductus arteriosus that is similar or identical to that in the Poodle. The relatives of dogs with PDA, particularly parents, offspring, and siblings, should be screened for evidence of PDA. Dogs with PDA should not be used for breeding, regardless of breed.

[Structural features of the smooth muscle cells in the medial layer of the bifurcation of fetal cerebral arteries].

OBJECTIVE: To study the structural and histogenetic features of the smooth muscle cells (SMCs) in the medial layer of the bifurcation of human cerebral arteries in fetus. METHOD: Segments of the cerebral arteries from the circle of Willis including the bifurcations of normal human fetuses were sectioned in 1 of the 3 orthogonal planes, wherein the three- dimensional medial layer structure composed of SMCs was observed with light microscope. RESULT: Microscopic observation identified 1.1 to 9.0 SMC layers in the medial layer of the cerebral arteries from the circle of Willis in the fetuses at different weeks before birth, and 30.3% of the specimens were found to have focal defects of the medial SMC layers at the bifurcation of the developing arteries. CONCLUSION: The number of SMC layers consisted in the medial layers of the cerebral arteries develops with the fetal growth, and the identification of congenital anomaly of the medial SMCs may help verify the hypothetical pathogenesis for cerebral saccular aneurysms.