Magnetic Resonance Imaging-negative Acute Inflammatory Myelopathy following Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

A 55-year-old woman was admitted to our hospital because of gait disturbance and urinary retention that acutely emerged 1 week after severe acute respiratory syndrome coronavirus 2 infection. Acute inflammatory myelopathy was clinically suspected, based on bilateral lower-limb weakness with an extensor plantar response and an elevated immunoglobulin G level in the cerebrospinal fluid. Whole-spine magnetic resonance imaging findings were normal. The central conduction time was extended, based on somatosensory evoked potentials. Her lower-limb weakness was partially ameliorated with immunosuppressive therapy. Postinfectious myelopathy is a rare neurological complication of coronavirus disease 2019 and can develop with normal radiological findings.

HIF-2 directly activates CD82 gene expression in endothelial cells.

Hypoxia inducible factor (HIF)-1 and HIF-2 are transcription factors that mediate the cellular response to hypoxia. Although HIF-1 and HIF-2 share the same target genes, both proteins activate a distinct subset of genes. To identify the target genes preferentially activated by HIF-2 in endothelial cells, DNA microarray analysis was performed to human umbilical vein endothelial cells (HUVECs) with forced expression of either HIF-1α or HIF-2α. In the present study, which is the first comparative study of target genes induced by either HIF-1 or HIF-2 in HUVECs, HIF-1 (and not HIF-2) stimulated mainly glycolytic, hexose metabolic and alcohol metabolic gene expression. However, HIF-2 (but not HIF-1) induced developmental gene expressions such as Fms-like tyrosine kinase 1 (Flt-1) and angiopoietin 2 (Angpt2). Furthermore, CD82 was up-regulated by HIF-2, but not by HIF-1, in response to hypoxia. HIF-2 regulated CD82 gene expression by binding to its HRE consensus sequence located within its first intron. Assessing the function of CD82 in HUVECs forced its expression. This result revealed that CD82 negatively regulates the HUVECs cell migration. The induction of CD82 gene expression in endothelial cells provided new insights into a specific function of HIF-2.

FHL-2 suppresses VEGF-induced phosphatidylinositol 3-kinase/Akt activation via interaction with sphingosine kinase-1.

OBJECTIVE: In the functional screening of a human heart cDNA library to identify a novel antiangiogenic factor, the prime candidate gene was "four-and-a-half LIM only protein-2" (FHL-2). The goal of this study is to clear the mechanism of antiangiogenic signaling of FHL-2 in endothelial cells (ECs). METHODS AND RESULTS: Overexpressed FHL-2 strongly inhibited vascular endothelial growth factor (VEGF)-induced EC migration. In the angiogenic signaling, we focused on sphingosine kinase-1 (SK1), which produces sphingosine-1-phosphate (S1P), a bioactive sphingolipid, as a potent angiogenic mediator in ECs. Immunoprecipitation and immunostaining analysis showed that FHL-2 might bind to SK1. Importantly, overexpression of FHL-2 in ECs inhibited VEGF-induced SK1 activity, phosphatidylinositol 3-kinase activity, and phosphorylation of Akt and eNOS. In contrast, overexpression of FHL-2 had no effect on S1P-induced Akt phosphorylation. Interestingly, VEGF stimulation decreased the binding of FHL-2 and SK1. Depletion of FHL-2 by siRNA increased EC migration accompanied with SK1 and Akt activation, and increased the expression of VEGF receptor-2 which further enhanced VEGF signaling. Furthermore, injection of FHL-2 mRNA into Xenopus embryos resulted in inhibition of vascular network development, assessed by in situ hybridization with endothelial markers. CONCLUSIONS: FHL-2 may regulate phosphatidylinositol 3-kinase/Akt via direct suppression of the SK1-S1P pathway in ECs.

Tie2 is tied at the cell-cell contacts and to extracellular matrix by angiopoietin-1.

Angiopoietin-1 (Ang1) binds to and activates Tie2 receptor tyrosine kinase. Ang1-Tie2 signal has been proposed to exhibit two opposite roles in the controlling blood vessels. One is vascular stabilization and the other is vascular angiogenesis. There has been no answer to the question as to how Tie2 induces two opposite responses to the same ligand. Our group and Dr. Alitalos group have demonstrated that trans-associated Tie2 at cell-cell contacts and extracellular matrix (ECM)-anchored Tie2 play distinct roles in the endothelial cells. The complex formation depends on the presence or absence of cell-cell adhesion. Here, we review how Ang1-Tie2 signal regulates vascular maintenance and angiogenesis. We further point to the unanswered questions that must be clarified to extend our knowledge of vascular biology and to progress basic knowledge to the treatment of the diseases in which Ang1-Tie2-mediated signal is central.

HIF-1alpha signaling upstream of NKX2.5 is required for cardiac development in Xenopus.

HIF-1alpha is originally identified as a transcription factor that activates gene expression in response to hypoxia. In metazoans, HIF-1alpha functions as a master regulator of oxygen homeostasis and regulates adaptive responses to change in oxygen tension during embryogenesis, tissue ischemia, and tumorigenesis. Because Hif-1alpha-deficient mice exhibit a number of developmental defects, the precise role of HIF-1alpha in early cardiac morphogenesis has been uncertain. Therefore, to clarify the role of HIF-1alpha in heart development, we investigated the effect of knockdown of HIF-1alpha in Xenopus embryos using antisense morpholino oligonucleotide microinjection techniques. Knockdown of HIF-1alpha resulted in defects of cardiogenesis. Whole mount in situ hybridization for cardiac troponin I (cTnI) showed the two separated populations of cardiomyocytes, which is indicative of cardia bifida, in HIF-1alpha-depleted embryos. Furthermore, the depletion of HIF-1alpha led to the reduction in cTnI expression, suggesting the correlation between HIF-1alpha and cardiac differentiation. We further examined the expression of several heart markers, nkx2.5, gata4, tbx5, bmp4, hand1, and hand2 in HIF-1alpha-depleted embryos. Among them, the expression of nkx2.5 was significantly reduced. Luciferase reporter assay using the Nkx2.5 promoter showed that knockdown of HIF-1alpha decreased its promoter activity. The cardiac abnormality in the HIF-1alpha-depleted embryo was restored with co-injection of nkx2.5 mRNA. Collectively, these findings reveal that HIF-1alpha-regulated nkx2.5 expression is required for heart development in Xenopus.

Constitutive over-expression of VEGF results in reduced expression of Hand-1 during cardiac development in Xenopus.

During heart development, various signaling cascades are tightly regulated in a stage- and region-dependent manner. Vascular endothelial growth factor (VEGF) is one of the important molecules required for both vascular development and cardiac morphogenesis. VEGF receptors are present in the embryonic heart, so we focused on heart formation in VEGF-over-expressing Xenopus embryos. Over-expression of VEGF(170) caused disorganized vessels, while the expression of an endothelial marker, Tie-2, was increased. The embryo's heart was distinctly larger than that of control, and showed abnormal morphology. Histological analysis of these embryos showed failure of heart looping. In situ hybridization with Hand-1, which controls intrinsic morphogenetic pathways, revealed that the expression level of Hand-1 was decreased in the heart region. These results suggest that increased VEGF(170) levels disturb Hand-1 expression in the region required for normal heart morphogenesis. VEGF expression level may be important in heart morphology during embryonic development.

Novel mechanisms of valsartan on the treatment of acute myocardial infarction through inhibition of the antiadhesion molecule periostin.

Our previous study demonstrated that periostin, an extracellular matrix protein, plays an important role in left ventricular remodeling through the inhibition of cell-cell interactions. Because the gene regulation of periostin has not yet been examined, we focused on the effects of angiotensin (Ang) II and mechanical stretch, because Ang II and mechanical stretch are related to cardiac remodeling after myocardial infarction. First, we examined the effects of Ang II on periostin in myocytes and fibroblasts in vitro. Ang II significantly increased periostin through phosphatidylinositol 3-kinase, c-Jun N-terminal kinase, p38, and extracellular signal-regulated kinase 1/2 pathways in myocytes and fibroblasts (P<0.05). On the other hand, mechanical stretch also significantly increased periostin expression (P<0.05). This increase was inhibited partially, but significantly, by an Ang II receptor blocker, valsartan, and inhibited almost completely by valsartan with the neutralization antibodies for transforming growth factor-beta and platelet-derived growth factor-BB (P<0.05). Therefore, we further examined periostin expression in vivo. Periostin expression was significantly increased in infarcted myocardium (P<0.05), and treatment with valsartan significantly attenuated it at 4 weeks after myocardial infarction (P<0.05), accompanied by a significant improvement in cardiac dysfunction (P<0.05). Overall, the present study demonstrated that Ang II, as well as mechanical stretch, stimulated periostin expression in both cardiac myocytes and fibroblasts, whereas valsartan significantly attenuated the increase in periostin expression. The inhibition of periostin by valsartan might especially contribute to its beneficial effects on cardiac remodeling after myocardial infarction.

The effect of VEGF on blood vessels and blood cells during Xenopus development.

Vascular endothelial growth factor (VEGF) is known to play an essential role in vascular development. We have overexpressed VEGF122 or VEGF170, which are equivalent to mouse VEGF120 and VEGF164, in developing Xenopus embryos. Overexpression of VEGF170 but not VEGF122 demonstrated an absence of expression of hematopoietic markers alpha-globin and GATA-1 but only in the posterior portion of the blood island. Interestingly, strong signals of endothelial markers, msr, fli-1, and tie-2, were detectable in those regions, instead of hematopoietic markers. These results suggested both that injection of VEGF170 resulted in disturbance of vasculogenesis in the posterior portion of the blood island, with excessive production of endothelial cells at the expense of blood cells, and that the anterior and posterior portions of the VBI may have distinct characteristics.

Hypoxic up-regulation of triosephosphate isomerase expression in mouse brain capillary endothelial cells.

A protein with a molecular mass of 27kDa was induced by hypoxia in a mouse brain capillary endothelial cell line and identified as triosephosphate isomerase (TPI) by amino-terminal sequencing. Hypoxia caused an elevation of the TPI protein level, concomitant with an increase of the TPI mRNA level. However, hypoxia resulted in an insufficient elevation of TPI activity level, compared to an increase of TPI protein level. When cells expressing the recombinant TPI protein with histidine tag were exposed to hypoxia and the TPI protein was affinity-purified, the catalytic activity (specific activity) of the TPI protein purified from hypoxic cells was substantially lower than that obtained from normoxic cells. In addition, three TPI isoforms with an electrophoretic multiplicity were found; two of the three isoforms were substantially increased in response to the hypoxia, but the level of the most acidic isoform was barely changed. The induction of TPI gene expression by hypoxia was suppressed by (1) a chelator of intracellular Ca(2+), (2) a blocker of non-selective cation channels, (3) a blocker of Na(+)/Ca(2+) exchangers, (4) an inhibitor of Ca(2+)/calmodulin-dependent protein kinases, and (5) an inhibitor of c-jun/AP-1 activation.

Increased expression of CYR61, an extracellular matrix signaling protein, in human benign prostatic hyperplasia and its regulation by lysophosphatidic acid.

Lysophosphatidic acid (LPA) is an endogenous lipid growth factor that is thought to play important roles in cell proliferation and antiapoptosis and therefore may have roles in the development and progression of benign prostatic hyperplasia (BPH). CYR61 (CCN1), on the other hand, is a growth factor-inducible immediate early gene that functions in cell proliferation, differentiation, and extracellular matrix synthesis. Here we show the close relationship between LPA-induced expression of CYR61 and prostate enlargement. CYR61 mRNA and protein were dramatically up-regulated by 18:1 LPA (oleoyl-LPA) within 1 and 2 h, respectively, in both stromal and epithelial prostatic cells. G protein-coupled receptors, i.e. Edg-2, Edg-4, and Edg-7, for LPA were also expressed in both stromal and epithelial prostatic cells. Furthermore, on DNA microarray analysis for normal and BPH patients, CYR61 was found to be related to the development and progression of BPH, regardless of symptoms. Although CYR61 mRNA was synthesized in hyperplastic epithelial cells, in many cases of BPH, CYR61 protein was detected in both the epithelial and stromal regions of BPH patient tissues. The functional contribution of CYR61 to prostatic cell growth was demonstrated by recombinant CYR61 protein and anti-CYR61 neutralizing antibodies, which inhibited CYR61-dependent cell spreading and significantly diminished cell proliferation, respectively. In conclusion, these data support the hypothesis that LPAs induce the expression of CYR61 by activating G proteincoupled receptors and that CYR61 acts as a secreted autocrine and/or paracrine mediator in stromal and epithelial hyperplasia, demonstrating the potential importance of this signaling mechanism in the disease.

Bordetella dermonecrotic toxin undergoes proteolytic processing to be translocated from a dynamin-related endosome into the cytoplasm in an acidification-independent manner.

Bordetella pertussis dermonecrotic toxin (DNT), which activates intracellular Rho GTPases, is a single chain polypeptide composed of an N-terminal receptor-binding domain and a C-terminal enzymatic domain. We found that DNT was cleaved by furin, a mammalian endoprotease, on the C-terminal side of Arg(44), which generates an N-terminal fragment almost corresponding to the receptor-binding domain and a C-terminal remainder (deltaB) containing the enzymatic domain. These two fragments remained associated even after the cleavage and made a nicked form. DNT mutants insensitive to furin had no cellular effect, whereas the nicked toxin was much more potent than the intact form, indicating that the nicking by furin was a prerequisite for action. DeltaB, but not the nicked toxin, associated with artificial liposomes and activated Rho in cells resistant to DNT because of a lack of surface receptor. These results imply that deltaB, dissociated from the binding domain, fully possesses the ability to enter the cytoplasm across the lipid bilayer membrane. The translocation ability of deltaB was found to be attributable to the N-terminal region encompassing amino acids 45-166, including a putative transmembrane domain. Pharmacological analyses with various reagents disturbing vesicular trafficking revealed that the translocation requires neither the acidification of the endosomes nor retrograde vesicular transport to deeper organelles, although DNT appeared to be internalized via a dynamin-dependent endocytosis. We conclude that DNT binds to its receptor and is internalized into endosomes where the proteolytic processing occurs. DeltaB, liberated from the binding domain after the processing, begins to translocate the enzymatic domain into the cytoplasm.

Nitric oxide synthase induction, cGMP elevation, and biopterin synthesis in vascular smooth muscle cells stimulated with interleukin-1beta in hypoxia.

In cultured rat vascular smooth muscle cells (VSMC), inducible nitric oxide synthase (iNOS) expression evoked by interleukin-1beta (IL-1beta) or tumor necrosis factor-alpha was greatly enhanced in hypoxia (2% O(2)), compared to in normoxia. In contrast, iNOS induction by interferon-gamma, lipopolysaccharide or their combination was barely influenced by hypoxia. These results indicate that iNOS induction is regulated by hypoxia in different manners, depending on the stimuli in VSMC. Nitric oxide (NO) production in response to stimulation with interferon-gamma plus lipopolysaccharide was significantly decreased in hypoxia, due to a decrease in the concentration of O(2) as a substrate. In contrast, the level of NO production in hypoxia was almost the same as that in normoxia when the cells were stimulated by IL-1beta. In addition, cGMP increased in response to IL-1beta in hypoxia to a level comparable to that in normoxia. Thus, it seems that the IL-1beta-induced expression of iNOS is up-regulated in hypoxia to compensate for a decrease in the enzyme activity due to the lower availability of O(2) as a substrate, and consequently a sufficient amount of NO is produced to elevate cGMP to an adequate level. In addition, the IL-1beta-induced synthesis of tetrahydrobiopterin, a cofactor for iNOS, was also greatly stimulated by hypoxia in VSMC.

Hypoxia up-regulates glyceraldehyde-3-phosphate dehydrogenase in mouse brain capillary endothelial cells: involvement of Na+/Ca2+ exchanger.

The molecular regulatory mechanisms and the characterization of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in hypoxia were studied in a mouse brain capillary endothelial cell line, MBEC4. Activation of GAPDH gene expression by hypoxia was suppressed by an intracellular Ca(2+) chelator and inhibited by a non-selective cation channel blocker or a Na(+)/Ca(2+) exchanger (NCX) blocker. Sequencing of reverse transcription-PCR products demonstrated that MBEC4 expressed an mRNA encoding NCX3, which functions even under cellular ATP-depleted conditions, in addition to mRNAs encoding NCX1 and NCX2. The inhibition of Ca(2+)/calmodulin-dependent protein kinases or c-Jun/AP-1 activation caused a significant decrease in the activation of GAPDH mRNA by hypoxia. These results suggest that hypoxia stimulates Ca(2+) influx through non-selective cation channels and causes the reverse operation of the three NCX isoforms, and consequently, increased intracellular Ca(2+) up-regulates GAPDH gene expression through an AP-1-dependent pathway. Furthermore, subcellular fractionation experiments showed that hypoxia increased GAPDH proteins not only in the cytosolic fraction, but also in the nuclear and particulate fractions, in which GAPDH should play no roles in glycolysis. However, the GAPDH activity did not rise in proportion to the increase of GAPDH protein by hypoxia even in the cytosolic fraction. These results suggest that not all hypoxia-induced GAPDH molecules contribute to glycolysis.

Association of Pasteurella multocida toxin with vimentin.

To help understand the molecular mechanisms of Pasteurella multocida toxin (PMT) action, we searched for a cellular protein interacting with PMT. The ligand overlay assay revealed a 60-kDa cellular protein that binds to a region from the 840th to 985th amino acids of the toxin. This protein was identified as vimentin by peptide mass fingerprinting. The N-terminal head domain of vimentin was further found to be responsible for the binding to the toxin.