PHD1, PHD2, and PHD3 are prolyl hydroxylase domain proteins that regulate the stability of hypoxiainducible factor ␣ subunits (HIF-␣). To determine the roles of individual PHDs during mouse development, we disrupted all three Phd genes and found that Phd2 ؊/؊ embryos died between embryonic days 12.5 and 14.5 whereas Phd1؊/؊ or Phd3 ؊/؊ mice were apparently normal. In Phd2 ؊/؊ mice, severe placental and heart defects preceded embryonic death. Placental defects included significantly reduced labyrinthine branching morphogenesis, widespread penetration of the labyrinth by spongiotrophoblasts, and abnormal distribution of trophoblast giant cells. The expression of several trophoblast markers was also altered, including an increase in the spongiotrophoblast marker Mash2 and decreases in the labyrinthine markers Tfeb and Gcm1. In the heart, trabeculae were poorly developed, the myocardium was remarkably thinner, and interventricular septum was incompletely formed. Surprisingly, while there were significant global increases in HIF-␣ protein levels in the placenta and the embryo proper, there was no specific HIF-␣ increase in the heart. Taken together, these data indicate that among all three PHD proteins, PHD2 is uniquely essential during mouse embryogenesis.

Oxygen-dependent hydroxylation of hypoxia-inducible factor (HIF)-a subunits by prolyl hydroxylase domain (PHD) proteins signals their polyubiquitination and proteasomal degradation, and plays a critical role in regulating HIF abundance and oxygen homeostasis. While oxygen concentration plays a major role in determining the efficiency of PHD-catalyzed hydroxylation reactions, many other environmental and intracellular factors also significantly modulate PHD activities. In addition, PHDs may also employ hydroxylase-independent mechanisms to modify HIF activity. Interestingly, while PHDs regulate HIF-a protein stability, PHD2 and PHD3 themselves are subject to feedback upregulation by HIFs. Functionally, different PHD isoforms may differentially contribute to specific pathophysiological processes, including angiogenesis, erythropoiesis, tumorigenesis, and cell growth, differentiation and survival. Because of diverse roles of PHDs in many different processes, loss of PHD expression or function triggers multi-faceted pathophysiological changes as has been shown in mice lacking different PHD isoforms. Future investigations are needed to explore in vivo specificity of PHDs over different HIF-a subunits and differential roles of PHD isoforms in different biological processes.

Polycythemia is often associated with erythropoietin (EPO) overexpression and defective oxygen sensing. In normal cells, intracellular oxygen concentrations are directly sensed by prolyl hydroxylase domain (PHD)-containing proteins, which tag hypoxia-inducible factor (HIF) ␣ subunits for polyubiquitination and proteasomal degradation by oxygen-dependent prolyl hydroxylation. Here we show that different PHD isoforms differentially regulate HIF-␣ stability in the adult liver and kidney and suppress Epo expression and erythropoiesis through distinct mechanisms. Although Phd1 ؊/؊ or Phd3 ؊/؊ mice had no apparent defects, double knockout of Phd1 and Phd3 led to moderate erythrocytosis. HIF-2␣, which is known to activate Epo expression, accumulated in the liver. In adult mice deficient for PHD2, the prototypic Epo transcriptional activator HIF-1␣ accumulated in both the kidney and liver. Elevated HIF-1␣ levels were associated with dramatically increased concentrations of both Epo mRNA in the kidney and Epo protein in the serum, which led to severe erythrocytosis. In contrast, heterozygous mutation of Phd2 had no detectable effects on blood homeostasis. These findings suggest that PHD1/3 double deficiency leads to erythrocytosis partly by activating the hepatic HIF-2␣/Epo pathway, whereas PHD2 deficiency leads to erythrocytosis by activating the renal Epo pathway. (Blood. 2008; 111:3229-3235)

Erythropoietin (Epo) is produced by renal Epo-producing cells (REPs) in a hypoxia-inducible manner. The conversion of REPs into myofibroblasts and coincident loss of Epo-producing ability are the major cause of renal fibrosis and anemia. However, the hypoxic response of these transformed myofibroblasts remains unclear. Here, we used complementary in vivo transgenic and live imaging approaches to better understand the importance of hypoxia signaling in Epo production. Live imaging of REPs in transgenic mice expressing green fluorescent protein from a modified Epo-gene locus revealed that healthy REPs tightly associated with endothelium by wrapping processes around capillaries. However, this association was hampered in states of renal injury-induced inflammation previously shown to correlate with the transition to myofibroblast-transformed renal Epo-producing cells (MF-REPs). Furthermore, activation of hypoxia-inducible factors (HIFs) by genetic inactivation of HIF-prolyl hydroxylases (PHD1, PHD2, and PHD3) selectively in Epo-producing cells reactivated Epo production in MF-REPs. Loss of PHD2 in REPs restored Epo-gene expression in injured kidneys but caused polycythemia. Notably, combined deletions of PHD1 and PHD3 prevented loss of Epo expression without provoking polycythemia. Mice with PHD-deficient REPs also showed resistance to LPS-induced Epo repression in kidneys, suggesting that augmented HIF signaling counterbalances inflammatory stimuli in regulation of Epo production. Thus, augmentation of HIF signaling may be an attractive therapeutic strategy for treating renal anemia by reactivating Epo synthesis in MF-REPs.

Functional near-infrared spectroscopy (fNIRS) is used to measure cerebral activity because it is simple and portable. However, scalp-hemodynamics often contaminates fNIRS signals, leading to detection of cortical activity in regions that are actually inactive. Methods for removing these artifacts using standard source-detector distance channels (Long-channel) tend to over-estimate the artifacts, while methods using additional short source-detector distance channels (Short-channel) require numerous probes to cover broad cortical areas, which leads to a high cost and prolonged experimental time. Here, we propose a new method that effectively combines the existing techniques, preserving the accuracy of estimating cerebral activity and avoiding the disadvantages inherent when applying the techniques individually. Our new method accomplishes this by estimating a global scalp-hemodynamic component from a small number of Short-channels, and removing its influence from the Long-channels using a general linear model (GLM). To demonstrate the feasibility of this method, we collected fNIRS and functional magnetic resonance imaging (fMRI) measurements during a motor task. First, we measured changes in oxygenated hemoglobin concentration (∆Oxy-Hb) from 18 Short-channels placed over motor-related areas, and confirmed that the majority of scalp-hemodynamics was globally consistent and could be estimated from as few as four Short-channels using principal component analysis. We then measured ∆Oxy-Hb from 4 Short- and 43 Long-channels. The GLM identified cerebral activity comparable to that measured separately by fMRI, even when scalp-hemodynamics exhibited substantial task-related modulation. These results suggest that combining measurements from four Short-channels with a GLM provides robust estimation of cerebral activity at a low cost.

Background-Prolyl hydroxylase domain (PHD) proteins, including PHD1, PHD2, and PHD3, mediate oxygen-dependent degradation of hypoxia-inducible factor (HIF)-␣ subunits. Although angiogenic roles of hypoxia-inducible factors are well known, the roles of PHDs in the vascular system remain to be established. Methods and Results-We evaluated angiogenic phenotypes in mice carrying targeted disruptions in genes encoding different PHD isoforms. Although Phd1 Ϫ/Ϫ and Phd3 Ϫ/Ϫ mice did not display apparent angiogenic defects, broadspectrum conditional knockout of Phd2 led to hyperactive angiogenesis and angiectasia. Blood vessels in PHD2-deficient mice were highly perfusable. Furthermore, examination of medium-sized vessels in subendocardial layer in the heart demonstrated successful recruitment of vascular smooth muscle cells. Surprisingly, increased vascular growth was independent of local efficiency of Phd2 disruption. Mice carrying significant Phd2 disruption in multiple organs, including the liver, heart, kidney, and lung, displayed excessive vascular growth not only in these organs but also in the brain, where Phd2 disruption was very inefficient. More surprisingly, increased accumulation of hypoxia-inducible factor-1␣ and angiectasia in the liver were not accompanied by corresponding increases in hepatic expression of Vegfa or angiopoietin-1. However, the serum vascular endothelial growth factor-A level was significantly increased in PHD2-deficient mice. Conclusions-PHD2, but not PHD1 and PHD3, is a major negative regulator for vascular growth in adult mice. Increased angiogenesis in PHD2-deficient mice may be mediated by a novel systemic mechanism. (Circulation. 2007;116:774-781.)

Anodal tDCS transiently enhanced knee extensor strength. The modest increase was specific to the LL. Thus, tDCS might augment the rehabilitation of stroke patients when combined with lower extremity strengthening or functional training.

Abstract-Recently, it was shown that Rho-kinase plays an important role in blood pressure regulation. However, it is not known whether Rho-kinase is involved in atherogenesis. Monocyte chemoattractant protein-1 (MCP-1) is an important chemokine that regulates monocyte recruitment and atherogenesis. Therefore, we examined the role of Rho and Rho-kinase in the angiotensin (Ang) II-induced expression of MCP-1. Ang II dose-and time-dependently enhanced the expression of MCP-1 mRNA and the protein production in vascular smooth muscle cells. CV11974, an Ang II type 1 receptor (AT 1 -R) specific antagonist inhibited the enhancement of MCP-1 expression by Ang II, suggesting that the effect of Ang II is mediated by the AT 1 -R. Botulinum C3 exotoxin, a specific inhibitor of Rho, suppressed Ang II-induced MCP-1 production. Key Words: angiotensin II Ⅲ peptides Ⅲ muscle, smooth, vascular Ⅲ receptors, angiotensin Ⅲ kinase A ngiotensin (Ang) II has been known to regulate blood pressure, fluid homeostasis, and electrolyte balance. 1 Recent studies have shown that Ang II plays an important role in atherogenesis as well. The physiological functions of Ang II are transmitted into target cells through its specific receptor located in the plasma membrane. Although there are 2 isoforms for the Ang II receptor, the Ang II type 1 receptor (AT 1 -R) 2 and the Ang II type 2 receptor (AT 2 -R), 3 most of the cardiovascular effects of Ang II are ascribed to AT 1 -R. Vascular smooth muscle cells (VSMCs) express AT 1 -R, and Ang II induces the production of growth factors and extracellular matrices through this receptor. We have recently reported that Ang II induced interleukin-6 production in VSMCs and have proposed that the Ang II-induced cytokine plays an important role in the progression of atherosclerosis. 4 Invasion of monocytes into the blood vessel wall is one of the early steps in the development of atherosclerosis. Various cytokines, such as monocyte chemoattractant protein-1 (MCP-1), 5 macrophage inflammatory protein-1, 6 and RANTES (regulated on activation, normal T-expressed and -secreted), 7 are known to regulate the movement of monocytes. Among these factors, MCP-1 is one of the most potent chemoattractants for monocytes or macrophages in vitro and in vivo. MCP-1 expression is induced in response to tumor necrosis factor-␣ or ␥-interferon, 8 thrombin, 9 or interleukin-1␤ 10 in VSMCs. Pathological conditions such as hypercholesterolemia and vascular injury also induce expression of the MCP-1 gene in the vascular wall. Recent studies have suggested that MCP-1 is critical for the progression of atherosclerosis. Targeted disruption of the receptor for MCP-1 (CCR2) attenuated the development of atherosclerosis when the mice were crossed with apoE knockout mice that develop severe atherosclerosis. 11 Rho-kinase, identified as a downstream target of Rho A, has been shown to phosphorylate the myosin-binding subunit of myosin light chain phosphatase and enhance smooth muscle contraction. 12,13 Y-27632, a specific inhibitor of Rho-kina...