Calcineurin inhibitors exacerbate coronary arteritis via the MyD88 signalling pathway in a murine model of Kawasaki disease.

Calcineurin inhibitors (CNIs) have been used off-label for the treatment of refractory Kawasaki disease (KD). However, it remains unknown whether CNIs show protective effects against the development of coronary artery lesions in KD patients. To investigate the effects of CNIs on coronary arteries and the mechanisms of their actions on coronary arteritis in a mouse model of KD, we performed experiments with FK565, a ligand of nucleotide-binding oligomerization domain-containing protein 1 (NOD1) in wild-type, severe combined immunodeficiency (SCID), caspase-associated recruitment domain 9 (CARD9)-/- and myeloid differentiation primary response gene 88 (MyD88)-/- mice. We also performed in-vitro studies with vascular and monocytic cells and vascular tissues. A histopathological analysis showed that both cyclosporin A and tacrolimus exacerbated the NOD1-mediated coronary arteritis in a dose-dependent manner. Cyclosporin A induced the exacerbation of coronary arteritis in mice only in high doses, while tacrolimus exacerbated it within the therapeutic range in humans. Similar effects were obtained in SCID and CARD9-/- mice but not in MyD88-/- mice. CNIs enhanced the expression of adhesion molecules by endothelial cells and the cytokine secretion by monocytic cells in our KD model. These data indicated that both vascular and monocytic cells were involved in the exacerbation of coronary arteritis. Activation of MyD88-dependent inflammatory signals in both vascular cells and macrophages appears to contribute to their adverse effects. Particular attention should be paid to the development of coronary artery lesions when using CNIs to treat refractory KD.

Forensic identification using multiple lot numbers of an implanted device.

We report a case in which identification of a deceased individual was established using multiple lot numbers printed on a body implantable device. Autopsy of an unknown woman revealed an intramedullary nail inserted within her right femur. The device manufacturer was identified from the configuration of the intramedullary nail, and the "use history" was traced from lot numbers printed on the device's multiple parts. The deceased individual was thus identified as a woman who had attempted suicide by jumping from a height about a year previously and had been transported to a hospital and undergone surgery that included implantation of the intramedullary nail. The main factor contributing to the rapid identification was the manufacturer's and distributor's record of the use history (traceability) of the product, because of their accountability for purposes of quality control. A second contributing factor was multiple lot numbers, resulting in extremely low probability of the same combination of lot numbers being present in multiple individuals. This case confirmed the utility of multiple lot numbers of body implantable devices in forensic identification.

Hindered proton collectivity in 16(28)S12: possible magic number at Z=16.

The reduced transition probability B(E2;0(gs)(+)→2(1)(+)) for (28)S was obtained experimentally using Coulomb excitation at 53 MeV/nucleon. The resultant B(E2) value 181(31) e(2)fm(4) is smaller than the expectation based on empirical B(E2) systematics. The double ratio |M(n)/M(p)|/(N/Z) of the 0(gs)(+)→2(1)(+) transition in (28)S was determined to be 1.9(2) by evaluating the M(n) value from the known B(E2) value of the mirror nucleus (28)Mg, showing the hindrance of proton collectivity relative to that of neutrons. These results indicate the emergence of the magic number Z=16 in the |T(z)|=2 nucleus (28)S.

Counting-backward test for executive function in idiopathic normal pressure hydrocephalus.

OBJECTIVES: The aim of this study was to develop and validate a bedside test for executive function in patients with idiopathic normal pressure hydrocephalus (INPH). MATERIALS AND METHODS: Twenty consecutive patients with INPH and 20 patients with Alzheimer's disease (AD) were enrolled in this study. We developed the counting-backward test for evaluating executive function in patients with INPH. Two indices that are considered to be reflective of the attention deficits and response suppression underlying executive dysfunction in INPH were calculated: the first-error score and the reverse-effect index. Performance on both the counting-backward test and standard neuropsychological tests for executive function was assessed in INPH and AD patients. RESULTS: The first-error score, reverse-effect index and the scores from the standard neuropsychological tests for executive function were significantly lower for individuals in the INPH group than in the AD group. The two indices for the counting-backward test in the INPH group were strongly correlated with the total scores for Frontal Assessment Battery and Phonemic Verbal Fluency. The first-error score was also significantly correlated with the error rate of the Stroop colour-word test and the score of the go/no-go test. In addition, we found that the first-error score highly distinguished patients with INPH from those with AD using these tests. CONCLUSION: The counting-backward test is useful for evaluating executive dysfunction in INPH and for differentiating between INPH and AD patients. In particular, the first-error score may reflect deficits in the response suppression related to executive dysfunction in INPH.

Two patients with focal segmental glomerulosclerosis complicated by cyclosporine-induced reversible posterior leukoencephalopathy syndrome.

Reversible posterior leukoencephalopathy syndrome (RPLS) is a distinctive clinicoradiological entity observed in a variety of clinical settings. Cyclosporine (CyA)-RPLS has been reported in a few patients with focal segmental glomerulosclerosis (FSGS); however, there had been no reports on developed RPLS after the re-administration of CyA treatment. We report two patients with FSGS who developed CyA-induced RPLS and summarize the results of a literature review for similar patients. The two patients with FSGS presented here were a 4-year-old boy and a 9-year-old boy, who presented with steroid-resistant nephrotic syndrome (NS) and were treated with CyA. The first patient developed CyA-induced RPLS at the 7th day after the start of CyA treatment, and the second patient at the 16th day after the re-start of CyA treatment. The two patients complained of a visual disorder and exhibited signs of a disturbance in consciousness and hypertension. Electroencephalography (EEG) examinations revealed a generalized slow wave pattern, and magnetic resonance imaging (MRI) disclosed an area of high signal intensity in the white matter. Subsequently, CyA was discontinued and neurological symptoms improved and recrudescence of RPLS did not occur. Our findings suggest that patients with FSGS and NS who are treated with CyA should be closely monitored for the possible onset of RPLS, presenting as a disturbance in consciousness, visual disturbances and/or convulsions.

Relationship between metabolic syndrome components and vascular properties in Japanese type 2 diabetic patients without cardiovascular disease or nephropathy.

To investigate the effect of metabolic syndrome (MS) components on early atherosclerosis markers, i.e., urinary albumin excretion rate (UAE), pulse wave velocity (PWV), and carotid intima-media thickness (IMT), we studied 536 Japanese patients with type 2 diabetes without cardiovascular disease or nephropathy. The MS definition by ATP III was employed. UAE, PWV, and IMT increased significantly with increasing the number of components even before fulfilling the diagnosis of MS. UAE was significantly influenced by high blood pressure, high triglycerides, and low HDL cholesterol. PWV was significantly increased by high blood pressure. IMT was significantly increased by high blood pressure and abdominal obesity. Multiple regression analysis, including MS components and putative risk factors, indicated that the number of MS components, age and glycosylated HbA1C were independent determinants of UAE, PWV, and IMT. LDL cholesterol and male gender were independent determinants of IMT. In conclusion, UAE, PWV, and IMT increased according to increasing the number of MS in type 2 diabetic patients without cardiovascular disease or diabetic nephropathy. The current observation considering the modifiable factors may help to identify patients who are at high risk of experiencing cardiovascular disease.

DNA Polymerase Beta Participates in Mitochondrial DNA Repair.

We have detected DNA polymerase beta (Polß), known as a key nuclear base excision repair (BER) protein, in mitochondrial protein extracts derived from mammalian tissue and cells. Manipulation of the N-terminal sequence affected the amount of Polß in the mitochondria. Using Polß fragments, mitochondrion-specific protein partners were identified, with the interactors functioning mainly in DNA maintenance and mitochondrial import. Of particular interest was the identification of the proteins TWINKLE, SSBP1, and TFAM, all of which are mitochondrion-specific DNA effectors and are known to function in the nucleoid. Polß directly interacted functionally with the mitochondrial helicase TWINKLE. Human kidney cells with Polß knockout (KO) had higher endogenous mitochondrial DNA (mtDNA) damage. Mitochondrial extracts derived from heterozygous Polß mouse tissue and KO cells had lower nucleotide incorporation activity. Mouse-derived Polß null fibroblasts had severely affected metabolic parameters. Indeed, gene knockout of Polß caused mitochondrial dysfunction, including reduced membrane potential and mitochondrial content. We show that Polß is a mitochondrial polymerase involved in mtDNA maintenance and is required for mitochondrial homeostasis.

Influence of ABCB1 C3435T polymorphism on the pharmacokinetics of lansoprazole and gastroesophageal symptoms in Japanese renal transplant recipients classified as CYP2C19 extensive metabolizers and treated with tacrolimus.

OBJECTIVE: Lansoprazole and tacrolimus are substrates of ATP binding cassette (ABC) transporters such as P-glycoprotein (ABCBI/multidrug resistance 1) and cytochrome P450 (CYP). The purpose of this study was to investigate the implication of the ABCB1 C3435Tpolymorphism on the pharmacokinetics of (R)-lansoprazole, the major enantiomer, in CYP2C19 extensive metabolizers (EMs) and on gastroesophageal symptoms in renal transplant recipients receiving tacrolimus. MATERIALS: 24 recipients who were CYP2C19 EMs were studied. METHODS: Oral administration of 30 mg lansoprazole was started 2 days before transplantation. On Day 2 before and Day 28 after transplantation, the plasma concentrations of (R)-lansoprazole and tacrolimus were measured. RESULTS: Pretransplantation, there were no significant differences in the pharmacokinetic parameters of (R)-lansoprazole between the 3 ABCBI C3435T genotypes. However, after renal transplantation, the peak plasma concentration (Cma ) and area under the plasma concentration-time curve (AUCO-24) of (R)-lansoprazole in patients with the ABCB1 C3435T C allele significantly increased, but not in patients with the TT genotype. These pharmacokinetic variations in (R)-lansoprazole did not influence the AUC of tacrolimus. There were no significant differences in the frequency of gastroesophageal symptoms among the three ABCB] C3435Tgenotypes. CONCLUSIONS: (R)-lansoprazole concentrations significantly increased in CYP2C19 extensive metabolizers with the ABCB1 C3435T C allele, but not TT genotype, after renal transplantation. However, the clinical relevance of this observation may be minor because these pharmacogenetic changes were not associated with the occurrence of gastroesophageal complications.

Repair of apurinic/apyrimidinic sites by UV damage endonuclease; a repair protein for UV and oxidative damage.

UV damage endonuclease (UVDE) initiates a novel form of excision repair by introducing a nick imme-diately 5" to UV-induced cyclobutane pyrimidine dimers or 6-4 photoproducts. Here, we report that apurinic/apyrimidinic (AP) sites are also nicked by Neurospora crassa and Schizosaccharomyces pombe UVDE. UVDE introduces a nick immediately 5" to the AP site leaving a 3"-OH and a 5"-phosphate AP. Apyrimidinic sites are more effectively nicked by UVDE than apurinic sites. UVDE also possesses 3"-repair activities for AP sites nicked by AP lyase and for 3"-phosphoglycolate produced by bleomycin. The Uvde gene introduced into Escherichia coli cells lacking two types of AP endonuclease, Exo III and Endo IV, gave the host cells resistance to methylmethane sulfonate and t-butyl hydroperoxide. We identified two AP endonuclease activities in S.pombe cell extracts. Besides cyclobutane pyrimidine dimers and 6-4 photoproducts, N. crassa UVDE also nicks Dewar photoproducts. Thus, UVDE is able to repair both of the major forms of DNA damage in living organisms: UV-induced DNA lesions and AP sites.

Roles of two VEGF receptors, Flt-1 and KDR, in the signal transduction of VEGF effects in human vascular endothelial cells.

Vascular endothelial growth factor (VEGF) is a principal regulator of vasculogenesis and angiogenesis. VEGF expresses its effects by binding to two VEGF receptors, Flt-1 and KDR. However, properties of Flt-1 and KDR in the signal transduction of VEGF-mediated effects in endothelial cells (ECs) were not entirely clarified. We investigated this issue by using two newly developed blocking monoclonal antibodies (mAbs) against Flt-1 and KDR. VEGF elicits DNA synthesis and cell migration of human umbilical vein endothelial cells (HUVECs). The pattern of inhibition of these effects by two mAbs indicates that DNA synthesis is preferentially mediated by KDR. In contrast, the regulation of cell migration by VEGF appears to be more complicated. Flt-1 regulates cell migration through modulating actin reorganization, which is essential for cell motility. A distinct signal is generated by KDR, which influences cell migration by regulating cell adhesion via the assembly of vinculin in focal adhesion plaque and tyrosine-phosphorylation of focal adhesion kinase (FAK) and paxillin.

Maturation of nascent DNA fragment is disturbed after treatment of cultured mouse FM3A cells with 8-methoxypsoralen plus near-ultraviolet radiation.

By the method of sedimentation in 5-20% alkaline sucrose gradient, the process of maturation of the nascent DNA fragment was studied with cultured mouse FM3A cells treated with 8-methoxypsoralen plus near-ultraviolet radiation. This treatment is known to cause crosslinks of the chromosomal DNA strands. The profile of the newly-replicated DNA, labeled for 10 min with [3H]thymidine immediately after treatment, was the same as that of the untreated cells, where the incorporated radioactivity was present in the intermediate DNA fragment (about 50-80 S). But, when the treated cells were labeled after several hours of incubation, the labeled DNA became much shorter due to inhibition of maturation of the initial DNA fragment (the Okazaki fragment) to the intermediate DNA. With the use of aphidicolin, a specific inhibitor of eukaryotic DNA polymerase alpha, it became apparent that, in addition to formation of the crosslinks, further DNA replication is required to cause this inhibition of DNA maturation. Aphidicolin also suppressed the inhibition of incorporation of [3H]thymidine into cellular DNA after treatment, but inhibition of this incorporation resumed after its removal.

Establishment of a simple and practical procedure applicable to therapeutic angiogenesis.

BACKGROUND: Therapeutic angiogenesis is thought to be beneficial for serious ischemic diseases. This investigation was designed to establish a simple and practical procedure applicable to therapeutic angiogenesis. METHODS AND RESULTS: When cultured skeletal muscle cells were electrically stimulated at a voltage that did not cause their contraction, vascular endothelial growth factor (VEGF) mRNA was augmented at an optimal-frequency stimulation. This increase of VEGF mRNA was derived primarily from transcriptional activation. Electrical stimulation increased the secretion of VEGF protein into the medium. This conditioned medium then augmented the growth of endothelial cells. The effect of electrical stimulation was further confirmed in a rat model of hindlimb ischemia. The tibialis anterior muscle in the ischemic limb was electrically stimulated. The frequency of stimulation was 50 Hz and strength was 0.1 V, which was far below the threshold for muscle contraction. After a 5-day stimulation, there was a significant increase in blood flow within the muscle. Immunohistochemical analysis revealed that VEGF protein was synthesized and capillary density was significantly increased in the stimulated muscle. Rats tolerated this procedure very well, and there was no muscle contraction, muscle injury, or restriction in movement. CONCLUSIONS: We propose this procedure as a simple and practical method of therapeutic angiogenesis.

Angiotensin-converting enzyme inhibitor preserves p21 and endothelial nitric oxide synthase expression in monocrotaline-induced pulmonary arterial hypertension in rats.

BACKGROUND: Pulmonary arterial hypertension (PAH) is associated with structural changes in the pulmonary vasculature characterized by the proliferation of cellular components of the vessels. ACE inhibitor (ACEI) may have beneficial effects in treating PAH, but its precise mechanism of action in the remodeling process is unclear. p21 is a cyclin-dependent kinase inhibitor that may have a protective role in this process by inhibiting cellular proliferation. Endothelial nitric oxide synthase (eNOS) has also been shown to be protective by its vasodilatory effect. Therefore, we investigated whether expression of p21 and eNOS was modulated by ACEI treatment in a rat model. METHODS AND RESULTS: Monocrotaline (MCT) was administered to 2 groups of Sprague-Dawley rats fed a high-cholesterol diet, ie, one group received MCT concomitantly with enalapril treatment (MCT(+)/ACEI(+) rats), and the other group did not receive enalapril (MCT(+)/ACEI(-) rats). After 5 weeks, MRI showed right ventricular hypertrophy in MCT(+)/ACEI(-) rats. MCT(+)/ACEI(+) rats showed a preserved right ventricular morphology. Isolated pulmonary perfusion studies showed that ACEI significantly upregulated NO production, as measured by nitrite levels. Addition of N-methyl-D-glucamine dithiocarbamate-Fe solution, an NO-trapping agent, reversed the basal vasodilatory effect of ACEI in the pulmonary vasculature. Immunoblot analysis showed decreased p21 and eNOS expression in the lung in MCT(+)/ACEI(-) rats, whereas their expression was preserved with enalapril treatment. CONCLUSIONS: ACEI suppresses the development of MCT-induced PAH in rats. The mechanism of action might involve the preservation of p21 and eNOS expression. Both p21 and endothelium-derived NO appear to have protective roles in the development of PAH.

Attenuation of myocardial ischemia/reperfusion injury by superinduction of inducible nitric oxide synthase.

BACKGROUND: Nitric oxide (NO) has been implicated as a mediator in myocardial ischemia/reperfusion (I/R) injury, but its functional properties have been conflicting. We investigated whether NO has a protective role against I/R injury. METHODS AND RESULTS: Using endothelial NO synthase knockout (eNOS KO) mice, inducible NOS KO mice, the NO donor S-nitroso-N-acetylpenicillamine (SNAP), and the NOS inhibitor N-iminoethyl-L-ornithine (L-NIO), we performed studies of isolated perfused hearts subjected to 30 minutes of global ischemia followed by reperfusion. After 60 minutes of reperfusion, nitrite levels in the coronary effluent in the SNAP and eNOS KO groups were significantly elevated compared with other groups. Immunoblot and immunohistochemistry showed that iNOS was markedly induced in the eNOS KO hearts. Under spontaneous beating conditions during reperfusion, increased NO activity was correlated with a prevention of the hyperdynamic contractile response and enhanced myocardial protection, as evidenced by a reduction in myocardial injury and infarct size. During prolonged reperfusion, SNAP-treated hearts were able to preserve contractile functions for 180 minutes, whereas L-NIO-treated hearts showed a sustained deterioration in contractility. CONCLUSIONS: NO protects against I/R injury by preventing the hyperdynamic response of isolated perfused hearts during early reperfusion. In the eNOS KO hearts, a paradoxical increase in NO production was seen, accompanied by a superinduction of iNOS, possibly due to an adaptive mechanism.

Macrophage accumulation associated with rat cardiac allograft rejection detected by magnetic resonance imaging with ultrasmall superparamagnetic iron oxide particles.

BACKGROUND: Acute cardiac allograft rejection continues to be the cause of graft loss and contributes to the morbidity and mortality after cardiac transplantation. In this study, we report a new method for detecting organ rejection in transplantation with an MR-based technique using dextran-coated ultrasmall superparamagnetic iron oxide (USPIO) particles. These particles ( approximately 27 nm in diameter) are known to shorten relaxation times in MRI experiments. METHODS AND RESULTS: A new rat model of heterotopic heart and lung transplantation has been developed for MRI experiments. Allotransplantations (DA-->BN) were performed (n=8), with syngeneic transplantations (BN-->BN) serving as controls (n=8). MR images were obtained with a gradient echo method. At postoperative day 7, allotransplants developed moderate rejection as determined histopathologically. A significant reduction in MR signal intensity was observed after USPIO injection into rats with allotransplanted hearts. Syngeneic transplants showed no differences in MR signal intensity before and after USPIO injections. After injection of USPIO particles at postoperative day 6, a group of allotransplanted rats was treated with cyclosporin A (3 mg/kg). Animals treated with cyclosporin A for 7 days showed no reduction in MR signal intensity after USPIO reinjection at day 14, whereas animals treated for 4 days showed a significant decrease in MR signal intensity in the transplanted hearts indicative of acute graft rejection. Pathological analysis of these animals revealed that dextran-coated USPIO particles were taken up by the infiltrating macrophages that accumulated within the rejecting cardiac graft. CONCLUSIONS: This MRI method offers promise as a noninvasive method for detecting transplant allograft rejection.

Oxidative damage to mitochondria is a preliminary step to caspase-3 activation in fluoride-induced apoptosis in HL-60 cells.

It has been suggested that oxidative stress plays a major role in various forms of cell death, including necrosis and apoptosis. We have previously reported that fluoride (NaF) induces apoptosis in HL-60 cells by caspase-3 activation. The main focus of this investigation was to arrive at a possible pathway of the apoptosis induced by NaF upstream of caspase-3, because the mechanism is still unknown. The present study showed that after exposure to NaF, there was an increase in MDA and 4-HNE and a loss of mitochondrial membrane potential (deltaPsi(m)) was also observed in NaF-treated cells. There was a significant increase in cytosolic cytochrome c, which is released from the mitochondria. We have reported a downregulation of Bcl-2 protein in NaF-treated cells. The antioxidants N-acetyl cysteine (NAC), glutathione (GSH) protected the cells from loss of deltaPsi(m), and there was no cytochrome c exit or Bcl-2 downregulation, and we suggest that these antioxidants prevent apoptosis induced by NaF. These results suggested that perhaps NaF induced apoptosis by oxidative stress-induced lipid peroxidation, causing loss of deltaPsi(m), and thereby releasing cytochrome c into the cytosol and further triggering the caspase cascade leading to apoptotic cell death in HL-60 cells.

Activation of c-Jun amino-terminal kinase is required for retinoic acid-induced neural differentiation of P19 embryonal carcinoma cells.

P19 embryonal carcinoma cells are known to differentiate into neurons and glia when treated with relatively high concentrations (>100 nM) of retinoic acid (RA). Concomitant with this RA-induced neural differentiation, we observed an activation of the c-Jun amino-terminal kinase (JNK). JNK was required for the RA-induced neural differentiation, because dominant-negative JNK blocked the differentiation. Studies using protein phosphatase inhibitors and protein kinase inhibitors suggested that both okadaic acid-sensitive protein phosphatase(s) and protein kinase C participate in the RA-induced activation of JNK.

A novel approach with magnetic resonance imaging used for the detection of lung allograft rejection.

OBJECTIVE: Although various techniques have been explored for the detection and quantification of allograft transplant rejection, a practical and reliable method that is noninvasive is still elusive. METHODS: For our magnetic resonance imaging experiments, we have developed a new rat model of heterotopic lung transplantation to the inguinal region. Allogeneic transplants (DA to Brown Norway) were performed with and without cyclosporine A (INN: ciclosporin) treatment, with syngeneic transplants (Brown Norway to Brown Norway) serving as controls (n = 6 per group). Magnetic resonance images were obtained with a gradient echo method before and after injection of ultra-small superparamagnetic iron oxide particles. RESULTS: At day 5, allogeneic transplants without cyclosporine A treatment showed a grade 4 rejection histologically. A significantly lower magnetic resonance signal was seen 24 hours after injection of ultra-small superparamagnetic iron oxide particles compared with the preinjection image (346 +/- 7.6 vs 839 +/- 43.4 arbitrary units; P <. 05). Syngeneic transplants showed no evidence of rejection histologically and no differences in magnetic resonance imaging signals between the images before and after injection of ultra-small superparamagnetic iron oxide particles (863 +/- 18.8 vs 880 +/- 22.5). Allotransplants treated with cyclosporine A showed a grade 2 rejection histologically. The change in magnetic resonance signals in that group was small but showed a significant decrease in signal intensity after injection (646 +/- 10.5 vs 889 +/- 23.5, P <.05). Immunohistochemistry and iron staining of the allografts indicated that ultra-small superparamagnetic iron oxide particles were taken up by the infiltrating macrophages that accumulated at the rejection site. CONCLUSIONS: We have demonstrated a novel approach for the detection of acute lung allograft rejection using magnetic resonance imaging coupled with injection of ultra-small superparamagnetic iron oxide particles. Despite its limitations, our method might be a first step toward a potential clinical application.

The role of myocardial gap junctions in electrical conduction and arrhythmogenesis.

Electrical activation of the heart requires cell-cell transfer of current via gap junctions, arrays of densely packed protein channels that permit intercellular passage of ions and small molecules. Because current transfer occurs only at gap junctions, the spatial distribution and biophysical properties of gap junction channels are important determinants of the conduction properties of cardiac muscle. Gap junction channels are composed of members of a multigene family of proteins called connexins. As a general rule, individual cells express multiple connexins, which creates the potential for considerable functional diversity in gap junction channels. Although gap junction channels are relatively nonselective in their permeability to ions and small molecules, cardiac myocytes actively adjust their level of coupling by multiple mechanisms including changes in connexin expression, regulation of connexin trafficking and turnover, and modulation of channel properties. In advanced stages of heart disease, connexin expression and intercellular coupling are diminished, and gap junction channels become redistributed. These changes have been strongly implicated in the pathogenesis of lethal ventricular arrhythmias. Ongoing studies in genetically engineered mice are revealing insights into the role of individual gap junction channel proteins in normal cardiac function and arrhythmogenesis.

Properties of two VEGF receptors, Flt-1 and KDR, in signal transduction.

The properties of two VEGF receptors, Flt-1 and KDR, in the signal transduction of VEGF in human umbilical vein endothelial cells (HUVECs) were investigated by using two newly developed blocking monoclonal antibodies (mAbs) against Flt-1 and KDR. VEGF stimulated the expression of transcription factor Ets-1 as well as matrix metalloproteinase-1 (MMP-1) and Flt-1 in HUVECs. The KDR/Flt-1 heterodimer and the KDR homodimer mediate the expression of Ets-1, MMP-1, and Flt-1. VEGF also stimulated DNA synthesis and migration of HUVECs. DNA synthesis is mediated by the same signaling system as the expression of Ets-1. In contrast, cell migration is regulated by two distinct signaling systems. The Flt-1 homodimer is required for actin reorganization. The KDR/Flt-1 heterodimer and the KDR homodimer are required for the assembly of vinculin in focal adhesion plaque by regulating the phosphorylation of focal adhesion kinase (FAK) and paxillin.