To examine whether the Janzen-Connell mechanism applies to temperate forests, seedling survival and causes of mortality were investigated at two distances (beneath, far) from conspecific adults and at two densities (high, low) at each distance for seedlings (n = 7935) of eight tree species co-occurring in a hardwood forest. Six of the eight species showed distance- and/or density-dependent seedling mortality mainly caused by diseases and rodents. In four of the five species primarily killed by disease (i.e. damping-off, blight, rot, powdery mildew), the infectivity (probability of infection by the disease) and/or the virulence (proportion of seedlings killed to those infected by the disease) were higher beneath than far from conspecific adults. These findings suggest that host specificity and/or spatially heterogeneous activity of natural enemies play an important role in the reciprocal replacement of tree species, maintaining species diversity in temperate forests.

Bone marrow- (BM-) derived cells can differentiate into smooth muscle-like cells (SMLC), resulting in vascular pathogenesis. However, the molecular mechanism of the differentiation remains unknown. We have recently reported that Notch signaling promotes while a Notch target HERP1 inhibit the differentiation of mesenchymal cells to SMC. During the differentiation of BM-derived mononuclear cells into smooth muscle α-actin (SMA)-positive cells, expression of Jagged1 and SMC-specific Notch3 was increased. Blocking Notch with γ-secretase inhibitor prevented the induction of SMA. Wire-mediated vascular injury was produced in femoral arteries in mice transplanted with green fluorescent protein (GFP)-positive cells. Many double-positive cells for GFP/Jagged1 or GFP/Notch3 were detected in the thickened neointima. In contrast, only a few SMA-positive cells were positive for GFP in neointima where HERP1, a suppressor for Notch, were abundantly expressed. In conclusion, Notch-HERP1 pathway plays an important role in differentiation of BM-derived mononuclear cells into SMLC.

Phenotypic plasticity and the switching of vascular smooth muscle cells (SMCs) play a critical role in atherosclerosis. Although Runx2, a key osteogenic transcription factor, is expressed in atherosclerotic plaques, the molecular mechanisms by which Runx2 regulates SMC differentiation remain unclear. Here we demonstrated that Runx2 repressed SMC differentiation induced by myocardin, which acts as a coactivator for serum response factor (SRF). Myocardin-mediated induction of SMC gene expression was enhanced in mouse embryonic fibroblasts derived from Runx2 null mice compared to wild-type mice. Forced expression of Runx2 decreased the expression of SMC genes and promoted osteogenic gene expression, whereas the reduction of Runx2 expression by small interfering RNA enhanced SMC differentiation in human aortic SMCs. Runx2 interacted with SRF and interfered with the formation of the SRF/myocardin ternary complex. Thus, this study provides the first evidence that Runx2 inhibits SRF-dependent transcription, as a corepressor independent of its DNA binding. We propose that Runx2 plays a pivotal role in osteogenic conversion tightly coupled with repression of the SMC phenotype in atherosclerotic lesions.

To examine whether the Janzen–Connell hypothesis is valid in temperate forests, we investigated the density, growth, mortality, and agents of mortality of seedlings and the density, size, and age of saplings of Prunus grayana Maxim. at three distances (0–3, 6–10, and 16–26 m) from conspecific adults in a temperate forest in Japan. An inoculation experiment was also conducted to test the host range of a leaf pathogen. The probability of mortality was highest at 0–3 m during the first 2 years of growth. Mortality mainly resulted from distance-dependent attack by two types of pathogen that caused damping-off epidemics and spot symptoms on leaves. The leaf pathogen was identified as Phaeoisariopsis pruni-grayanae Sawada, which infected many more seedlings of Prunus grayana than of the two other tree species tested in an inoculation experiment. The vertical and diameter growth was lowest at 0–3 m and highest at 16–26 m in both seedlings and saplings. As a result, the greatest number of large and older saplings was observed at 16–26 m. Our results demonstrate that the Janzen–Connell mechanism operates in a beech-dominated forest in the temperate region of Japan.

The Notch pathway is involved in multiple aspects of vascular development, including arterial-venous differentiation. Here, we show that Notch stimulation instructively induces arterial characteristics in endothelial cells (EC). Forced expression of Notch intracellular domain (NICD, activated form of Notch) induced mRNA expression for a subset of arterial-specific markers such as ephrinB2, connexin40, and HERP1 only in EC but not other cell lines. In co-culture experiments using EC and either Dll4- or Jagged1-expressing cells, we found that Dll4 stimulation but not Jagged1 markedly induced ephrinB2 expression. An inducible expression of HERP1 and HERP2 by NICD has no measurable effects on expression of ephrinB2 and venous marker EphB4 although either HERP1 or HERP2 overexpression exerts potent inhibitory effects on EphB4 expression without ephrinB2 induction. We also found no functional interaction between Notch and TGF-β–ALK1 signalings in an induction of ephrinB2 expression. These results suggest that Dll4-stimulated Notch signaling induces a part of arterial characteristics only in EC via HERP-independent mechanism. Our data provide new insight into the molecular mechanism of ligand-selective Notch activation during differentiation of arterial EC.

The Notch signaling pathway plays a crucial role in specifying cellular fates by interaction between cellular neighbors; however, the molecular mechanism underlying smooth muscle cell (SMC) differentiation by Notch signaling has not been well characterized. Here we demonstrate that Jagged1-Notch signaling promotes SMC differentiation from mesenchymal cells. Overexpression of the Notch intracellular domain, an activated form of Notch, up-regulates the expression of multiple SMC marker genes including SMC-myosin heavy chain (Sm-mhc) in mesenchymal 10T1/2 cells, but not in non-mesenchymal cells. Physiological Notch stimulation by its ligand Jagged1, but not Dll4, directly induces Sm-mhc expression in 10T1/2 cells without de novo protein synthesis, indicative of a ligand-selective effect. Jagged1-induced expression of SM-MHC was blocked byγ-secretase inhibitor, N-(N-(3,5-difluorophenyl)-l-alanyl)-S-phenylglycine t-butyl ester, which impedes Notch signaling. Using Rbp-jκ-deficient cells and site-specific mutagenesis of the SM-MHC gene, we show that such an induction is independent of the myocardin-serum response factor-CArG complex, but absolutely dependent on RBP-Jκ, a major mediator of Notch signaling, and its cognate binding sequence. Of importance, Notch signaling and myocardin synergistically activate SM-MHC gene expression. Taken together, these data suggest that the Jagged1-Notch pathway constitutes an instructive signal for SMC differentiation through an RBP-Jκ-dependent mechanism and augments gene expression mediated by the myocardin-SRF-CArG complex. Given that Notch pathway components are expressed in vascular SMC during normal development and disease, Notch signaling is likely to play a pivotal role in such situations to modulate the vascular smooth muscle cell phenotype.

Purpose: Human obesity is associated with abnormal accumulation of neutral lipid within metabolic organs and eventually leads to impaired function. Recent studies suggest that lipogenic gene, stearoyl-CoA deaturase-1 (SCD1), is up-regulated in skeletal muscle from obese humans. Although SCD1 is expressed in the heart, SCD1 involvement in cardiovascular disease is poorly understood. We therefore examined the expression of SCD1 in obese rat heart.

Objective— Myocardin is a coactivator of serum response factor (SRF) required for vascular smooth muscle cell (VSMC) differentiation. HERP1 is a transcriptional repressor, which is abundantly expressed in vascular system and is known to function as a target gene of Notch. However, the role of HERP1 in the pathogenesis of vascular lesions remains unknown. The present study characterizes the expression of HERP1 in normal and diseased vessels, and tests the hypothesis that HERP1 inhibits SRF/myocardin-dependent SMC gene expression. Methods and Results— Immunohistochemistry revealed that HERP1 and myocardin expression was localized to SMC in the neointima of balloon-injured rat aorta and in human coronary atherosclerotic lesions. Expression of both HERP1 and myocardin was elevated in cultured VSMCs compared with medial SMC. Overexpressed HERP1 inhibited the myocardin-induced SMC marker gene expression in 10T1/2 cells. HERP1 protein interfered with the SRF/CArG–box interaction in vivo and in vitro. Immunoprecipitation assays showed that HERP1 physically interacts with SRF. Conclusions— HERP1 expression was associated with the SMC proliferation and dedifferentiation in vitro and in vivo. HERP1 may play a role in promoting the phenotypic modulation of VSMCs during vascular injury and atherosclerotic process by interfering with SRF binding to CArG-box through physical association between HERP1 and SRF.

PURPOSEThe purpose of this study was to investigate the effect of the von Hippel-Lindau (VHL) protein on VEGF gene expression in vitro and to determine whether adenovirus-mediated VHL intraocular gene transfer inhibits the development of angiogenesis in a monkey model of multiple branch retinal vein occlusion (BRVO).METHODSA recombinant adenovirus vector adVHL was constructed to deliver the human VHL gene. Total RNA prepared from various kinds of cells transduced with adLacZ (control) or adVHL under normoxic or hypoxic conditions was subjected to Northern blot analyses. Either adLacZ or adVHL was delivered by preretinal injection in monkeys. The effects of adLacZ or adVHL on ocular neovascularization in laser-induced multiple BRVO was evaluated in color photographs and with fluorescein angiography (FA).RESULTSVHL expression in adVHL-transduced cells was confirmed at the transcript and protein levels. VHL overexpression significantly decreased the levels of VEGF transcripts in human aortic endothelial cells (HAECs); retinal pigment epithelium (RPE) cells; and RCC 786-O cells, renal carcinoma cells lacking VHL expression under normoxia. In contrast, VHL had no effect on the hypoxia-mediated increase in VEGF expression in these cells, although basal levels of VEGF expression were substantially reduced. Color photographs and FA revealed that retinal neovascularization and iris rubeosis accompanied by multiple BRVO in a monkey model were obviously suppressed by VHL overexpression. Northern blot analysis and immunostaining for VHL and VEGF indicated that VHL transfer obviously suppressed VEGF gene expression in VHL-transduced tissues such as retina or RPE.CONCLUSIONSThe results showed that adenovirus expressing VHL led to a significant reduction in VEGF expression in vitro under normoxic or hypoxic conditions. adVHL effectively inhibited angiogenesis in retina and iris in laser-induced multiple BRVO in monkey eyes. These data suggest that gene therapy based on VHL gene delivery has potential in the treatment of human ocular neovascularization.

Phospholamban (PLB) inhibits SR Ca(2+)-ATPase 2 (SERCA2) Ca(2+) uptake and is a potential therapeutic target in the context of heart failure. RNA interference (RNAi) is a technique that produces sequence-specific, post-transcriptional gene silencing through the use of double-stranded RNA directed against the homologous target gene. The goal of the current study was to investigate the efficacy of the RNAi method for ablation of PLB gene expression and restoration of Ca(2+) uptake function in cultured neonatal rat cardiac myocytes in which SERCA2 protein levels were decreased. Myocytes were transfected with 21-nucleotide duplexes of small interfering RNA (siRNA) targeting PLB (30 nmol/l) or with scramble sequence using a haemagglutinating virus of Japan (HVJ) envelope vector. Administration of PLB siRNA resulted in the reduction of PLB mRNA level to approximately 6% of that observed after administration of scramble siRNA group at 12 h after transfection. Further, PLB protein levels in the PLB siRNA groups were 12% of that in cells treated with scramble siRNA on day 2, and the mRNA and protein levels for SERCA2 and calsequestrin were not affected. In addition, Ca(2+) uptake affinity was increased in total homogenates from the PLB siRNA group (a 29% decrease in EC(50) value when compared with scramble siRNA group). Finally, PLB siRNA restored Ca(2+) uptake affinity following hydrogen peroxide-induced decreases in SERCA2 and PLB mRNA expression. These results demonstrate that PLB-targeted RNAi inhibited endogenous PLB expression in neonatal rat myocytes and restored Ca(2+) uptake affinity in cardiac myocytes in which SERCA2 protein levels were decreased. This technique may represent a novel therapeutic strategy for heart failure.