Primary Human Fibroblasts in Culture Switch to a Myofibroblast-Like Phenotype Independently of TGF Beta.

Fibroblasts are the prevalent cell type and main source for extracellular matrix (ECM) in connective tissue. Depending on their origin, fibroblasts play a central role in non-pathological tissue remodeling and disease like fibrosis. This study examined the effect of established culture conditions of primary human fibroblasts, from different origins on the myofibroblast-like phenotype formation. We isolated primary human fibroblasts from aortic adventitia, lung, juvenile- and adult skin and investigated the expression levels of CD90, alpha smooth muscle actin (αSMA) and procollagen I under different concentrations of fetal calf serum (FCS) and ascorbic acid (AA) in culture media by immunoblot and immunofluorescence assays. Furthermore, we determined the viability using XTT and migration/wound healing in scratch assays. Collagen 1 secretion was quantified by specific ELISA. Primary human fibroblasts show in part a myofibroblast-like phenotype even without addition of FCS. Supplemented AA reduces migration of cultured fibroblasts with no or low concentrations of FCS. Furthermore, AA and higher concentrations of FCS in culture media lead to higher levels of collagen 1 secretion instead of procollagen I accumulation. This study provides evidence for a partial switch of primary human fibroblasts of different origin to a myofibroblast-like phenotype under common culture conditions.

Extracellular matrix in ascending aortic aneurysms and dissections - What we learn from decellularization and scanning electron microscopy.

Pathological impairment of elastic fiber and other extracellular matrix (ECM) components are described for the aortic media of ascending thoracic aortic aneurysms (aTAA) but the exact pathological impairment of the structure and its degree still needs further investigations. To evaluate the quantity and quality of elastic fiber sheets and other ECM structures (e.g. collagen), cells were removed from different types of aneurysmal tissues (tricuspid aortic valve [TAV] associated-, bicuspid aortic valve [BAV] associated-aneurysmal tissue and acute aortic dissections [AAD]) using 2.5% sodium hydroxide (NaOH) and compared to decellularized control aortic tissue. Likewise, native tissue has been analysed. To evaluate the 2D- (histological evaluation, fluorescence- and auto-fluorescence based staining methods) and the 3D structure (scanning electron microscopic [SEM] examination) of the medial layer we first analysed for a successful decellularization. After proving for successful decellularization, we quantified the amount of elastic fiber sheets, elastin and other ECM components including collagen. Aside from clearly visible focal elastic fiber loss in TAV-aTAA tissue, decellularization resulted in reduction of elastic fiber auto-fluorescence properties, which is perhaps an indication from a disease-related qualitative impairment of elastic fibers, visible only after contact with the alkaline solution. Likewise, the loss of collagen amount in BAV-aTAA and TAV-aTAA tissue (compared to non-decellularized tissue) after contact with NaOH indicates a prior disease-associated impairment of collagen. Although the amount of ECM was not changed in type A dissection tissue, detailed electron microscopic evaluation revealed changes in ECM quality, which worsened after contact with alkaline solution but were not visible after histological analyses. Apart from the improved observation of the samples using electron microscopy, contact of aneurysmal and dissected tissue with the alkaline decellularization solution revealed potential disease related changes in ECM quality which can partly be connected to already published data, but have to be proven by further studies.

The megaaortic syndrome: Progression of ascending aortic aneurysm or a disease of distinct origin?

BACKGROUND: Thoracic aortic aneurysm (TAA) is an often asymptomatic disease with fatal outcome, such as dissection or rupture. The megaaortic syndrome (MAS) is an extensive dilatation of the whole aorta with low incidence but high lethal outcome with unknown pathophysiology so far. METHODS AND RESULTS: We compared aortic tissue of patients with sporadic TAAs and MAS of the ascending aorta with non-aneurysmal control tissues. Specimens of MAS patients showed a significantly reduced thickness of the media but an increased thickness of the intima compared to control tissue and TAAs with moderate dilatation. Advanced media degeneration however was detectable in both, TAAs with enhanced luminal diameter and MAS specimens, accompanied by reduced medial smooth muscle cell-density. Further specimens of MAS were characterized by massive atherosclerotic lesions in contrast to specimens of sporadic TAA patients. Infiltrations of macrophages in atherosclerotic lesions but also in the media adjacent to the adventitia were significantly elevated in tissue of TAAs with dilatation ≤6cm. Of note, atherosclerotic plaque-associated macrophages as well as those in the external media produce huge amounts of MMP-9 which is possibly involved in media degeneration and tissue destruction. CONCLUSIONS: Taken together these results demonstrate that the pathology of MAS shows similarities with that of TAAs but pathological differences in the ascending aorta, suggesting that MAS might be a disease of different origin.

Tissue engineering of heart valves: decellularized porcine and human valve scaffolds differ importantly in residual potential to attract monocytic cells.

BACKGROUND: Tissue-engineered or decellularized heart valves have already been implanted in humans or are currently approaching the clinical setting. The aim of this study was to examine the migratory response of human monocytic cells toward decellularized porcine and human heart valves, a pivotal step in the early immunologic reaction. METHODS AND RESULTS: Porcine and human pulmonary valve conduits were decellularized, and migration of U-937 monocytic cells toward extracted heart valve proteins was examined in a transmigration chamber in vitro. Homogenized tissue specimens were size fractionated by SDS-PAGE. The decellularization procedure effectively reduced the migration of human monocytes toward all heart valve tissue. However, only the antigen reduction of human pulmonary valves abolished the monocytic response (wall, 0.88+/-0.19% versus 30.20+/-3.93% migrated cells [mean+/-SEM]; cusps, 0.10+/-0.06% versus 10.24+/-1.83%) and was significantly lower (P<0.05) than that of the decellularized porcine equivalent (wall, 5.03+/-0.14% versus 24.31+/-2.38%; cusps, 3.18+/-0.38% versus 10.24+/-1.83%). SDS-PAGE of the pulmonary heart valve tissue revealed that considerable amounts of proteins with different molecular weights that were not detected in the human equivalent remain in the decellularized porcine heart valve. CONCLUSIONS: We describe for the first time that the remaining potential of decellularized pulmonary heart valves to attract monocytic cells depends strongly on whether porcine or human scaffolds were used. These findings will have an important impact on further investigations in the field of heart valve tissue engineering.

15-deoxy-delta12,14-prostaglandin-J2 inhibits expression of eNOS in human endothelial cells.

15-Deoxy-delta12,14-Prostaglandin-J2 (15d-PGJ2), an endogenous ligand of PPARgamma transcription factor, modifies expression of many genes involved in inflammation and angiogenesis. Enzyme which contributes to regulation of both these processes is endothelial nitric oxide synthase (eNOS). Our aim was to investigated the effect of 15d-PGJ2 on eNOS in human umbilical vein endothelial cells (HUVEC). We demonstrated that 24 h incubation of HUVEC with 15d-PGJ2 (1-10 microM) does not influence eNOS. On the contrary, the longer exposure (48-72 h) resulted in concentration-dependent inhibition of eNOS mRNA and protein expressions and led to reduction in eNOS enzymatic activity by approximately 50%. This effect was mediated by regulation of the transcription rate from eNOS promoter, what may be associated with inhibition of AP-1 binding capacity. The stability of mRNA was unchanged. Since none of the observed effects could be mimicked by troglitazone, a more potent PPARgamma ligand, we suppose that 15d-PGJ2 diminishes expression of eNOS via PPARgamma-independent mechanisms.