Parallel Murine and Human Plaque Proteomics Reveals Pathways of Plaque Rupture.

RATIONALE: Plaque rupture is the proximate cause of most myocardial infarctions and many strokes. However, the molecular mechanisms that precipitate plaque rupture are unknown. OBJECTIVE: By applying proteomic and bioinformatic approaches in mouse models of protease-induced plaque rupture and in ruptured human plaques, we aimed to illuminate biochemical pathways through which proteolysis causes plaque rupture and identify substrates that are cleaved in ruptured plaques. METHODS AND RESULTS: We performed shotgun proteomics analyses of aortas of transgenic mice with macrophage-specific overexpression of urokinase (SR-uPA+/0 mice) and of SR-uPA+/0 bone marrow transplant recipients, and we used bioinformatic tools to evaluate protein abundance and functional category enrichment in these aortas. In parallel, we performed shotgun proteomics and bioinformatics studies on extracts of ruptured and stable areas of freshly harvested human carotid plaques. We also applied a separate protein-analysis method (protein topography and migration analysis platform) to attempt to identify substrates and proteolytic fragments in mouse and human plaque extracts. Approximately 10% of extracted aortic proteins were reproducibly altered in SR-uPA+/0 aortas. Proteases, inflammatory signaling molecules, as well as proteins involved with cell adhesion, the cytoskeleton, and apoptosis, were increased. ECM (Extracellular matrix) proteins, including basement-membrane proteins, were decreased. Approximately 40% of proteins were altered in ruptured versus stable areas of human carotid plaques, including many of the same functional categories that were altered in SR-uPA+/0 aortas. Collagens were minimally altered in SR-uPA+/0 aortas and ruptured human plaques; however, several basement-membrane proteins were reduced in both SR-uPA+/0 aortas and ruptured human plaques. Protein topography and migration analysis platform did not detect robust increases in proteolytic fragments of ECM proteins in either setting. CONCLUSIONS: Parallel studies of SR-uPA+/0 mouse aortas and human plaques identify mechanisms that connect proteolysis with plaque rupture, including inflammation, basement-membrane protein loss, and apoptosis. Basement-membrane protein loss is a prominent feature of ruptured human plaques, suggesting a major role for basement-membrane proteins in maintaining plaque stability.

TGF-ß (Transforming Growth Factor-ß) Signaling Protects the Thoracic and Abdominal Aorta From Angiotensin II-Induced Pathology by Distinct Mechanisms.

OBJECTIVE: The role of TGF-ß (transforming growth factor-ß) signaling in abdominal aortic aneurysm (AAA) formation is controversial. Others reported that systemic blockade of TGF-ß by neutralizing antibodies accelerated AAA development in angiotensin II-infused mice. This result is consistent with other studies suggesting that TGF-ß signaling prevents AAA. Development of a therapy for AAA that exploits the protective actions of TGF-ß would be facilitated by identification of the mechanisms through which TGF-ß prevents AAA. We hypothesized that TGF-ß signaling prevents AAA by its actions on aortic medial smooth muscle cells. APPROACH AND RESULTS: We compared the prevalence, severity, and histopathology of angiotensin II-induced AAA among control mice (no TGF-ß blockade), mice with antibody-mediated systemic neutralization of TGF-ß, and mice with genetically based smooth muscle-specific loss of TGF-ß signaling. Surprisingly, we found that systemic-but not smooth muscle-specific-TGF-ß blockade significantly increased the prevalence of AAA and tended to increase AAA severity, adventitial thickening, and aortic wall macrophage accumulation. In contrast, abdominal aortas of mice with smooth muscle-specific loss of TGF-ß signaling differed from controls only in having a thinner media. We examined thoracic aortas of the same mice. Here we found that smooth muscle-specific loss of Tgfbr2-but not systemic TGF-ß neutralization-significantly accelerated development of aortic pathology, including increased prevalence of intramural hematomas, medial thinning, and adventitial thickening. CONCLUSION: Our results suggest that TGF-ß signaling prevents both abdominal and thoracic aneurysmal disease but does so by distinct mechanisms. Smooth muscle extrinsic signaling protects the abdominal aorta and smooth muscle intrinsic signaling protects the thoracic aorta.

Postnatal Deletion of the Type II Transforming Growth Factor-ß Receptor in Smooth Muscle Cells Causes Severe Aortopathy in Mice.

OBJECTIVE: Prenatal deletion of the type II transforming growth factor-ß (TGF-ß) receptor (TBRII) prevents normal vascular morphogenesis and smooth muscle cell (SMC) differentiation, causing embryonic death. The role of TBRII in adult SMC is less well studied. Clarification of this role has important clinical implications because TBRII deletion should ablate TGF-ß signaling, and blockade of TGF-ß signaling is envisioned as a treatment for human aortopathies. We hypothesized that postnatal loss of SMC TBRII would cause aortopathy. APPROACH AND RESULTS: We generated mice with either of 2 tamoxifen-inducible SMC-specific Cre (SMC-CreER(T2)) alleles and homozygous floxed Tgfbr2 alleles. Mice were injected with tamoxifen, and their aortas examined 4 and 14 weeks later. Both SMC-CreER(T2) alleles efficiently and specifically rearranged a floxed reporter gene and efficiently rearranged a floxed Tgfbr2 allele, resulting in loss of aortic medial TBRII protein. Loss of SMC TBRII caused severe aortopathy, including hemorrhage, ulceration, dissection, dilation, accumulation of macrophage markers, elastolysis, abnormal proteoglycan accumulation, and aberrant SMC gene expression. All areas of the aorta were affected, with the most severe pathology in the ascending aorta. Cre-mediated loss of SMC TBRII in vitro ablated both canonical and noncanonical TGF-ß signaling and reproduced some of the gene expression abnormalities detected in vivo. CONCLUSIONS: SMC TBRII plays a critical role in maintaining postnatal aortic homeostasis. Loss of SMC TBRII disrupts TGF-ß signaling, acutely alters SMC gene expression, and rapidly results in severe and durable aortopathy. These results suggest that pharmacological blockade of TGF-ß signaling in humans could cause aortic disease rather than prevent it.

Smooth muscle cells from abdominal aortic aneurysms are unique and can independently and synergistically degrade insoluble elastin.

BACKGROUND: The purpose of this study was to further elucidate the role of the vascular smooth muscle cells (SMCs) in abdominal aortic aneurysm (AAA) disease. We hypothesized that that AAA SMCs are unique and actively participate in the process of degrading the aortic matrix. METHODS: Whole-genome expression profiles of SMCs from AAAs, nondilated abdominal aorta (NAA), and carotid endarterectomy (CEA) were compared. We quantified elastolytic activity by culturing SMCs in [(3)H]elastin-coated plates and measuring solubilized tritium in the media after 7 days. Matrix metalloproteinase (MMP)-2 and MMP-9 production was assessed using real-time polymerase chain reaction, zymography, and Western blotting. RESULTS: Each SMC type exhibited a unique gene expression pattern. AAA SMCs had greater elastolytic activity than NAA-SMCs (+68%; P < .001) and CEA-SMCs (+45%; P < .001). Zymography showed an increase of active MMP-2 (62 kD) in media from AAA SMCs. AAA SMCs demonstrated twofold greater expression of MMP-2 messenger (m)RNA (P < .05) and 7.3-fold greater MMP-9 expression (P < .01) than NAA-SMCs. Culture with U937 monocytes caused a synergistic increase of elastolysis by AAA SMCs (41%; P < .001) but not NAA-SMCs or CEA-SMCs (P = .99). Coculture with U937 caused a large increase in MMP-9 mRNA in AAA-SMCs and NAA-SMCs (P < .001). MMP-2 mRNA expression was not affected. Western blots of culture media showed a fourfold increase of MMP-9 (92 kD) protein only in AAA-SMCs/U937 but not in NAA-SMCs/U937 (P < .001) and a large increase in active-MMP2 (62 kD), which was less apparent in NAA-SMCs/U937 media (P < .01). CONCLUSIONS: AAA-SMCs have a unique gene expression profile and a proelastolytic phenotype that is augmented by macrophages. This may occur by a failure of post-transcriptional control of MMP-9 synthesis.

Moderate aortic enlargement and bicuspid aortic valve are associated with aortic dissection in Turner syndrome: report of the international turner syndrome aortic dissection registry.

BACKGROUND: Girls and women with Turner syndrome are at risk for aortic dissection and rupture. However, the size of the aorta and the clinical characteristics among those with Turner syndrome and dissection have received little attention. METHODS AND RESULTS: We obtained medical records from 20 individuals who voluntarily participated in the International Turner Syndrome Aortic Dissection Registry. Type A dissections occurred in 17 of 20 (85%) cases, and type B occurred in 3 cases of which 1 occurred after coarctation stent placement. Of those with spontaneous aortic dissections, 18 of 19 (95%) had an associated cardiac malformation that included a bicuspid aortic valve. In 1 individual there was no predisposing finding other than the presence of Turner syndrome. Associated pregnancy was documented in 1 of 19 (5%). More than half (13/19, 68%) came to medical attention >24 hours after the onset of symptoms. For those with type A dissections, the mean ascending aortic size index was 2.7±0.6 cm/m(2) (n=9). CONCLUSIONS: Aortic dissection in Turner syndrome occurs in young individuals at smaller aortic diameters than in the general population or other forms of genetically triggered aortopathy. The absence of aortic valve or other cardiac malformations appears to markedly reduce the risk of aortic dissection However, aortic dissection can occur in Turner syndrome without cardiac malformations or hypertension. Individuals with Turner syndrome who are >18 years of age with an ascending aortic size index >2.5 cm/m(2) should be considered for an aortic operation to prevent aortic dissection.

Hypothermic Fibrillatory Arrest During Coronary Artery Bypass Grafting in a Man With Calcified Aorta and Ventricular Fibrillation.

A 67-year-old man undergoing coronary artery bypass grafting had aortic calcification that prohibited aortic cross-clamping. When ventricular fibrillation developed during surgery, we instituted hypothermic fibrillatory arrest to avoid aortic cross-clamping. In addition to our patient's case, we discuss the advantages and disadvantages of using hypothermic fibrillatory arrest during cardiac surgery.

Transanal presentation of a distal ventriculoperitoneal shunt catheter: Management of bowel perforation without laparotomy.

BACKGROUND: Bowel perforation is a serious but rare complication after a ventriculoperitoneal shunt (VPS) procedure. Prior studies have reported spontaneous bowel perforation after VPS placement in adults of up to 0.07%. Transanal catheter protrusion is a potential presentation of VPS bowel perforation and places a patient at risk for both peritonitis and ventriculitis/meningitis via retrograde migration of bacteria. This delayed complication can be fatal if unrecognized, with a 15% risk of mortality secondary to ventriculitis, peritonitis, or sepsis. CASE DESCRIPTION: We describe a unique case of a patient with distal VPS catheter protrusion from the anus whose bowel perforation did not cause clinical sequelae of infection. We were able to manage the patient without laparotomy. CONCLUSIONS: A subset of patients can be managed without laparotomy and only with externalization of the ventricular shunt with antibiotics until the cerebrospinal fluid cultures finalize without growth.

Frequency and outcomes of cardiac operations and catheter interventions in Turner syndrome.

Cardiac malformations occur commonly in Turner syndrome (TS), but the outcomes of cardiac operations and catheter-based procedures are unknown. The Pediatric Cardiac Care Consortium database was queried for individuals with TS and other female subjects without genetic abnormalities or syndromes (non-TS [NTS]). Procedures for left-sided heart lesions represented most TS procedures (95.2%). Three hundred ninety-eight patients with TS who underwent 637 of these procedures of interest were compared with 25,913 female NTS subjects who underwent 56,625 procedures. The numbers of procedures per admission (1.47 vs 1.61, p = 0.01) and per patient (1.85 vs 2.16, p <0.0001) were significantly lower in patients with TS. Procedures for cyanotic heart disease other than hypoplastic left heart (HLH) were performed 4.5-fold less frequently in patients with TS. Patients with TS and NTS subjects had equivalent hospital lengths of stay, except for patients with TS who underwent hypoplastic aortic arch operations, patent ductus arteriosus ligation, pulmonary artery balloon dilation, balloon atrial septostomy, and catheter closure of atrial septal defects. There were 34 deaths among patients with TS and 1,795 among NTS subjects (8.6% vs 7.2%, p = 0.30). When HLH was excluded, mortality was lower in the TS group (3.9% vs 6.5%, p = 0.05). Operations for partial anomalous pulmonary venous connection (14.3% vs 1.9%, p = 0.03) and HLH (90.4% vs 70.5%, p = 0.08) were more likely to result in death in patients with TS. In conclusion, given generally comparable lengths of stay and numbers of procedures as well as uniformly excellent results, these data suggest that the diagnosis of TS does not increase the utilization of limited health care resources. Operations for HLH and partial anomalous pulmonary vein connection carry additional risk for those with TS. These results will permit risk stratification, prognostication, and counseling of individuals with TS and their families.

Atrial natriuretic peptide induces natriuretic peptide receptor-cGMP-dependent protein kinase interaction.

Circulating natriuretic peptides such as atrial natriuretic peptide (ANP) counterbalance the effects of hypertension and inhibit cardiac hypertrophy by activating cGMP-dependent protein kinase (PKG). Natriuretic peptide binding to type I receptors (NPRA and NPRB) activates their intrinsic guanylyl cyclase activity, resulting in a rapid increase in cytosolic cGMP that subsequently activates PKG. Phosphorylation of the receptor by an unknown serine/threonine kinase is required before ligand binding can activate the cyclase. While searching for downstream PKG partners using a yeast two-hybrid screen of a human heart cDNA library, we unexpectedly found an upstream association with NPRA. PKG is a serine/threonine kinase capable of phosphorylating NPRA in vitro; however, regulation of NPRA by PKG has not been previously reported. Here we show that PKG is recruited to the plasma membrane following ANP treatment, an effect that can be blocked by pharmacological inhibition of PKG activation. Furthermore, PKG participates in a ligand-dependent gain-of-function loop that significantly increases the intrinsic cyclase activity of the receptor. PKG translocation is ANP-dependent but not nitric oxide-dependent. Our results suggest that anchoring of PKG to NPRA is a key event after ligand binding that determines distal effects. As such, the NPRA-PKG association may represent a novel mechanism for compartmentation of cGMP-mediated signaling and regulation of receptor sensitivity.