Myofibrillogenesis was studied in cultured chick cardiomyocytes using indirect immunofluorescence microscopy and antibodies against alpha- and gamma-actin, muscle and nonmuscle tropomyosin, muscle myosin, and titin. Initially, cardiomyocytes, devoid of myofibrils, developed variable numbers of stress fiber-like structures with uniform staining for anti-muscle and nonmuscle actin and tropomyosin, and diffuse, weak staining with anti-titin. Anti-myosin labeled bundles of filaments that exhibited variable degrees of association with the stress fiber-like structures. Myofibrillogenesis occurred with a progressive, and generally simultaneous, longitudinal reorganization of stress fiber-like structures to form primitive sarcomeric units. Titin appeared to attain its mature pattern before the other major contractile proteins. Changes in the staining patterns of actin, tropomyosin, and myosin as myofibrils matured were interpreted as due to longitudinal filament alignment occurring before ordering in the axial direction. Non-muscle actin and tropomyosin were found with sarcomeric periodicity in the initial stages of sarcomere myofibrillogenesis, although their staining patterns were not identical. The localization of the "sarcomeric" proteins alpha-actin and muscle tropomyosin in stress fiber-like structures and the incorporation of non-muscle proteins in the initial stages of sarcomere organization bring into question the meaning of "sarcomeric" proteins in regard to myofibrillogenesis.

Uremic patients have a much higher risk of cardiovascular diseases and death. Uremic toxins are probably involved in the development of vascular endothelial dysfunction. Indoxyl sulfate (IS) is a uremic toxin that accumulates with deterioration of renal function. This study explored the effects of IS on the adherens junctions of vascular endothelial cells and revealed the underlying mechanism. Bovine pulmonary artery endothelial cells (BPAECs) were treated with IS, and the distribution of vascular endothelial cadherin (VE-cadherin), p120-catenin, β-catenin, and stress fibers was examined by immunofluorescence. IS treatment resulted in disruption of intercellular contacts between BPAECs with prominent parallel-oriented intracellular stress fiber formation. Intracellular free radical levels which measured by flow cytometry increased after IS treatment. The antioxidant, MnTMPyP, and an ERK pathway inhibitor, U0126, both significantly prevented IS-induced disruption of intercellular contacts. Western blotting analyses demonstrated that IS-induced phosphorylation of myosin light chain kinase (MLCK) and myosin light chains (MLC) as well as activation of extracellular-signal-regulated protein kinase (ERK1/ERK2). Pretreatment with MnTMPyP prevented ERK1/2 phosphorylation. U0126 prevented the IS-induced MLCK and MLC phosphorylation. MEK-ERK acted as the upstream regulator of the MLCK-MLC pathway. These findings suggest that the superoxide anion-MEK-ERK-MLCK-MLC signaling mediates IS-induced junctional dispersal of BPAECs.

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Peripheral arterial diseases, the major complication of diabetes, can result in lower limb amputation. Since endothelial progenitor cells (EPCs) are involved in neovascularization, the aim of this study was to examine whether EPCs isolated from Wharton's jelly (WJ-EPCs) of the umbilical cord, a rich source of mesenchymal stem cells, could reduce ischemia-induced hind limb injury in diabetic mice. We evaluated the effects of WJ-EPC transplantation on hind limb injury caused by femoral artery ligation in mice with streptozotocin (STZ)-induced diabetes. We found that the ischemic hind limb in mice with STZ-induced diabetes showed decreased blood flow and capillary density and increased cell apoptosis and that these effects were significantly inhibited by an injection of WJ-EPCs. In addition, hypoxia-inducible factor-1a (HIF-1a) and interleukin-8 (IL-8) were highly expressed in transplanted WJ-EPCs in the ischemic skeletal tissues and were present at high levels in hypoxia-treated cultured WJ-EPCs. Moreover, incubation of the NOR skeletal muscle cell line under hypoxic conditions in conditioned medium from EPCs cultured for 16 h under hypoxic conditions resulted in decreased expression of pro-apoptotic proteins and increased expression of anti-apoptotic proteins. The inhibition of HIF-1a or IL-8 expression by EPCs using HIF-1a siRNA or IL-8 siRNA, respectively, prevented this change in expression of apoptotic-related proteins. Wharton's jelly in the umbilical cord is a valuable source of EPCs, and transplantation of these EPCs represents an innovative therapeutic strategy for treating diabetic ischemic tissues. The HIF-1a/IL-8 signaling pathway plays a critical role in the protective effects of EPCs in the ischemic hind limb of diabetic mice.

Cordyceps sinensis contains a factor that stimulates corticosteroid production in the animal model. However, it is not known whether this drug acts directly on the adrenal glands or indirectly via the hypothalamus-pituitary axis. In the present study, we used primary rat adrenal cell cultures to investigate the pharmacological function of a water-soluble extract of Cordyceps sinensis (CS) and the signaling pathway involved. Radioimmunoassay of corticosterone indicated that the amount of corticosterone produced by adrenal cells is increased in a positively dose-dependent manner by CS, reaching a maximum at 25 microg/ml. This stimulating effect was seen 1 h after CS treatment and was maintained for up to 24 h. Concomitantly, the lipid droplets in these cells became small and fewer in number. Immunostaining with a monoclonal antibody, A2, a specific marker for the lipid droplet capsule, demonstrated that detachment of the capsule from the lipid droplet occurs in response to CS application and that the period required for decapsulation is inversely related to the concentration of CS applied. The mechanism of CS-induced steroidogenesis is apparently different from that for ACTH, since intracellular cAMP levels were not increased in CS-treated cells. However, combined application with calphostin C, a PKC inhibitor, completely blocked the effect of CS on steroidogenesis, suggesting that activation of PKC may be responsible for the CS-induced steroidogenesis.