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Background: Marfan syndrome (MFS) is a heritable disorder of connective tissue, affecting principally skeletal, ocular, and cardiovascular systems. The most life-threatening manifestations are aortic aneurysm and dissection. We investigated changes in the proteome of aortic media in patients with and without MFS to gain insight into molecular mechanisms leading to aortic dilatation., Methods and Results: Aortic samples were collected from 46 patients. Twenty-two patients suffered from MFS, 9 patients had bicuspid aortic valve, and 15 patients without connective tissue disorder served as controls. Aortic media was isolated and its proteome was analyzed in 12 patients with the use of 2-dimensional difference gel electrophoresis and mass spectrometry. We found higher amounts of filamin A C-terminal fragment, calponin 1, vinculin, microfibril-associated glycoprotein 4, and myosin-10 heavy chain in aortic media of MFS aneurysm samples than in controls. Regulation of filamin A C-terminal fragmentation was validated in all patient samples by immunoblotting. Cleavage of filamin A and the calpain substrate spectrin was increased in the MFS and bicuspid aortic valve groups. Extent of cleavage correlated positively with calpain 2 expression and negatively with the expression of its endogenous inhibitor calpastatin., Conclusions: Our observation demonstrates for the first time upregulation of the C-terminal fragment of filamin A in dilated aortic media of MFS and bicuspid aortic valve patients. In addition, our results present evidence that the cleavage of filamin A is highly likely the result of the protease calpain. Increased calpain activity might explain, at least in part, histological alterations in dilated aorta.

The common carotid, vertebral, posterior cerebral arteries, and the aorta were studied in the human in terms of its eNOS expression. In around 10 weeks of gestation, the developing intima began to express notable eNOS. In the adult, the positive eNOS sites were in the endothelial cells and the tunica media where the smooth muscles were. In the vessels with athrosclerotic changes, eNOS was down regulated in the endothelial layer and most of the tunica media but was significantly upregulated in the tunica media around the lesion. The protein changes are related to the onset of the athrosclerotic diseases.

Although vascular cells express multiple members of the Nox family of nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase, including gp91phox, Nox1, and Nox4, the reasons for the different expressions and specific roles of these members in vascular injury in chronic hypertension have remained unclear. Thus, we quantified the mRNA expressions of these NAD(P)H oxidase components by real-time polymerase chain reaction and evaluated superoxide production and morphological changes in the aortas of 32-week-old stroke-prone spontaneously hypertensive rats (SHRSP) and age-matched Wistar Kyoto rats (WKY). The aortic media of SHRSP had an approximately 2.5-fold greater level of Nox4 mRNA and an approximately 10-fold greater level of Nox1 mRNA than WKY. The mRNA expressions of gp91phox and p22phox in SHRSP and WKY were comparable. SHRSP were treated from 24 weeks of age for 8 weeks with either high or low doses of candesartan (4 mg/kg/day or 0.2 mg/kg/day), or a combination of hydralazine (30 mg/kg/day) and hydrochlorothiazide (4.5 mg/kg/day). The high-dose candesartan or the hydralazine plus hydrochlorothiazide decreased the blood pressure of SHRSP to that of WKY, whereas the low-dose candesartan exerted no significant antihypertensive action. Media thickening and fibrosis, as well as the increased production of superoxide in SHRSP, were nearly normalized with high-dose candesartan and partially corrected with low-dose candesartan or hydralazine plus hydrochlorothiazide. These changes by antihypertensive treatment paralleled the decrease in mRNA expression of Nox4 and Nox1. These results suggest that blood pressure and angiotensin II type 1 receptor activation are involved in the up-regulation of Nox1 and Nox4 expression, which could contribute to vascular injury during chronic hypertension.

Background: Progressive medial degeneration and atrophy is thought to be a cause of ascending thoracic aortic aneurysms in the elderly. Extensive apoptosis of vascular smooth muscle cells (VSMCs) has been demonstrated in the media of abdominal aortic aneurysms. We investigated whether medial atrophy from loss of VSMCs occurs in primary ascending thoracic aortic aneurysms., Methods and Results: Morphometric analysis of 28 nonaneurysmal ascending thoracic aortas and 29 ascending thoracic aortic aneurysms was performed by directly measuring the thickness of their vascular layers and by indirectly calculating the area of their vascular compartments. The cellular and matrix composition of the media was assessed at the structural, protein, and transcript levels. Despite thinning of the media secondary to vascular dilatation, there was an overall increase in the medial area of aneurysms. VSMC density was preserved, implying cellular hyperplasia as a result of the increased medial mass. There was decreased expression of matrix proteins, despite sustained synthesis of these molecules, which was associated with evidence of increased matrix degradation. The remodeling and expansion of the media was most evident in comparisons between nonaneurysmal aortas versus smaller aneurysms and did not evolve further in larger aneurysms., Conclusions: The mechanisms for luminal enlargement in thoracic and abdominal aortic aneurysms differ significantly with regard to the survival of VSMCs and atrophy of the media but share common pathophysiology involving degeneration of the matrix. Hyperplastic cellular remodeling of the media in ascending thoracic aortic aneurysms may be an initial adaptive response to minimize increased wall stress resulting from vascular dilatation.

Here, we have found that type 2 iodothyronine deiodinase (D2) is present in rat aorta media and that there is a circadian variation in the D2 expression. The D2 mRNA was approximately 4-fold higher at 0900 h than at 2100 h, and the activity was approximately 6-fold higher at noon than at 2100 h. The increase in aorta media D2 activity is preceded by the increase in its mRNA. The increase in D2 mRNA and activity in the circadian variation was reduced by the administration of prazosin, an alpha1-adrenergic antagonist, and propranolol, a beta- adrenergic antagonist. Furthermore, phenylephrine, an alpha1-adrenergic agonist, and isoproterenol, a beta-adrenergic agonist, caused a significant increase in D2 mRNA and activity. In the hypothyroid rats, aorta mediae D2 mRNA at both 0900 and 2100 h were not significantly different when compared with those in the euthyroid rats. On the other hand, aorta mediae D2 activity at both 1200 and 2100 h in the hypothyroid rats were approximately 2-fold higher. From these results, we suggest that D2 activity of rat aorta media is increased by both alpha1- and beta-adrenergic stimulation, at least partly, at the pretranslational level. We also suggest that both alpha1- and beta-adrenergic mechanisms may be involved, at least partly, in the circadian variation of the activity. In the hypothyroid state, the aorta media D2 activity is increased mainly by the posttranslational mechanism, and the similar circadian variation of the D2 expression is present as in the euthyroid state.

The rat aorta, whose three wall layers can be separated by microdissection offers the rare possibility of comparing physiological characteristics of in vivo tissular cell components and corresponding cells after culture. We developed a technique allowing the dissociation of the three tunicae (intima, media and adventitia) of the rat aorta and the culture of their main cell types, i.e.: endothelial cells (EC) from intima, smooth muscle cells (SMC) from media and fibroblasts (Fib) from adventitia. Comparison between selected tunicae in vivo and their corresponding cells in vitro was performed via arterial angiotensin converting enzyme (ACE) activity measurements in Wistar rats. In vivo microsomial ACE activity for each tunica was as follows: 368.9 +/- 34.3 (endothelium), 10.5 +/- 1.9 (media) and 10.2 +/- 4.9 (adventitia) pmol/mg protein/min. Corresponding cell primary culture values were 1.2 +/- 0.1 (EC), 0.06 +/- 0.02 (SMC) and 0.24 +/- 0.01 (Fib) pmol/mg protein/min. Incubation of serum-deprived cells with Dexamethasone (10(-7) M) over 48 hr induced a statistically significant shift of total ACE activity from controls to stimulated cells of 2.9 +/- 0.3 to 9.7 +/- 1.0 in EC, 0.8 +/- 0.1 to 32.1 +/- 4.9 in SMC and 1.03 +/- 0.65 to 57.2 +/- 2.1 pmol/mg prot/min in fibroblasts. In the rat aorta, ACE was present not only in the intimal endothelial cell lining, but also in the media and the adventitia. ACE activity levels in primary cultured vascular cells were about 100-fold less than those found in the ex vivo tissues. Nevertheless, ACE expression seems to be more constitutive in endothelial cells and more inducible in smooth muscle cells and fibroblasts. This methodological approach should be of interest in studying environmental or genetic regulation of protein expression in the three layers/three cell types of the vascular wall.