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1.  Iron oxide-based nanomagnets in nanomedicine: fabrication and applications 
Nano Reviews  2010;1:10.3402/nano.v1i0.4883.
Iron oxide-based nanomagnets have attracted a great deal of attention in nanomedicine over the past decade. Down to the nanoscale, superparamagnetic iron oxide nanoparticles can only be magnetized in the presence of an external magnetic field, which makes them capable of forming stable colloids in a physio-biological medium. Their superparamagnetic property, together with other intrinsic properties, such as low cytotoxicity, colloidal stability, and bioactive molecule conjugation capability, makes such nanomagnets ideal in both in-vitro and in-vivo biomedical applications. In this review, a chemical, physical, and biological synthetic approach to prepare iron oxide-based nanomagnets with different physicochemical properties was illustrated and compared. The growing interest in iron oxide-based nanomagnets with multifunctionalities was explored in cancer diagnostics and treatment, focusing on their combined roles in a magnetic resonance contrast agent, hyperthermia, and magnetic force assisted drug delivery. Iron oxides as magnetic carriers in gene therapy were reviewed with a focus on the sophisticated design and construction of magnetic vectors. Finally, the iron oxide-based nanomagnet also represents a very promising tool in particle/cell interfacing in controlling cellular functionalities, such as adhesion, proliferation, differentiation, and cell patterning, in stem cell therapy and tissue engineering applications.
PMCID: PMC3215210  PMID: 22110854
iron oxide; coprecipitation; thermal decomposition; microemulsion; magnetosome; lithography; cancer targeting; stem cell; gene delivery; tissue engineering; cell actuation
2.  Selective ROCK2 Inhibition In Focal Cerebral Ischemia 
Rho-associated kinase (ROCK) is a key regulator of numerous processes in multiple cell types relevant in stroke pathophysiology. ROCK inhibitors have improved outcome in experimental models of acute ischemic or hemorrhagic stroke. However, the relevant ROCK isoform (ROCK1 or ROCK2) in acute stroke is not known.
We characterized the pharmacodynamic and pharmacokinetic profile, and tested the efficacy and safety of a novel selective ROCK2 inhibitor KD025 (formerly SLx-2119) in focal cerebral ischemia models in mice.
KD025 dose-dependently reduced infarct volume after transient middle cerebral artery occlusion. The therapeutic window was at least 3 hours from stroke onset, and the efficacy was sustained for at least 4 weeks. KD025 was at least as efficacious in aged, diabetic or female mice, as in normal adult males. Concurrent treatment with atorvastatin was safe, but not additive or synergistic. KD025 was also safe in a permanent ischemia model, albeit with diminished efficacy. As one mechanism of protection, KD025 improved cortical perfusion in a distal middle cerebral artery occlusion model, implicating enhanced collateral flow. Unlike isoform-nonselective ROCK inhibitors, KD025 did not cause significant hypotension, a dose-limiting side effect in acute ischemic stroke.
Altogether, these data show that KD025 is efficacious and safe in acute focal cerebral ischemia in mice, implicating ROCK2 as the relevant isoform in acute ischemic stroke. Data suggest that selective ROCK2 inhibition has a favorable safety profile to facilitate clinical translation.
PMCID: PMC3900310  PMID: 24466563
3.  Selective ROCK2 inhibition in focal cerebral ischemia 
Rho-associated kinase (ROCK) is a key regulator of numerous processes in multiple cell types relevant in stroke pathophysiology. ROCK inhibitors have improved outcome in experimental models of acute ischemic or hemorrhagic stroke. However, the relevant ROCK isoform (ROCK1 or ROCK2) in acute stroke is not known.
We characterized the pharmacodynamic and pharmacokinetic profile, and tested the efficacy and safety of a novel selective ROCK2 inhibitor KD025 (formerly SLx-2119) in focal cerebral ischemia models in mice.
KD025 dose-dependently reduced infarct volume after transient middle cerebral artery occlusion. The therapeutic window was at least 3 h from stroke onset, and the efficacy was sustained for at least 4 weeks. KD025 was at least as efficacious in aged, diabetic or female mice, as in normal adult males. Concurrent treatment with atorvastatin was safe, but not additive or synergistic. KD025 was also safe in a permanent ischemia model, albeit with diminished efficacy. As one mechanism of protection, KD025 improved cortical perfusion in a distal middle cerebral artery occlusion model, implicating enhanced collateral flow. Unlike isoform-nonselective ROCK inhibitors, KD025 did not cause significant hypotension, a dose-limiting side effect in acute ischemic stroke.
Altogether, these data show that KD025 is efficacious and safe in acute focal cerebral ischemia in mice, implicating ROCK2 as the relevant isoform in acute ischemic stroke. Data suggest that selective ROCK2 inhibition has a favorable safety profile to facilitate clinical translation.
PMCID: PMC3900310  PMID: 24466563
4.  Estimating the malaria transmission of Plasmodium vivax based on serodiagnosis 
Malaria Journal  2012;11:257.
Plasmodium vivax re-emerged in 1993 and has now become a major public health problem during the summer season in South Korea. The aim of this study was to interpret and understand the meaning of seroepidemiological studies for developing the best malaria control programme in South Korea.
Blood samples were collected in Gimpo city, Paju city, Yeoncheon County, Cheorwon County and Goseong County of high risk area in South Korea. Microscopy was performed to identify patients infected with P. vivax. Antibody detection for P. vivax was performed using indirect fluorescent antibody test (IFAT).
A total of 1,574 blood samples was collected from participants in the study areas and evaluated against three parameters: IFAT positive rate, annual antibody positive index (AAPI), and annual parasite index (API). The IFAT positive rate was 7.24% (n = 114). Of the five study areas, Gimpo had the highest IFAT positive rate (13.68%) and AAPI (4.63). Yeongcheon had the highest API in 2005 (2.06) while Gimpo had the highest API in 2006 (5.00). No correlation was observed between any of the three parameters and study sites' distance from the demilitarized zone (DMZ).
These results showed that P. vivax antibody levels could provide useful information about the prevalence of malaria in endemic areas. Furthermore, AAPI results for each year showed a closer relationship to API the following year than the API of the same year and thus could be helpful in predicting malaria transmission risks.
PMCID: PMC3470937  PMID: 22852558
5.  Striatal neurons expressing full-length mutant huntingtin exhibit decreased N-cadherin and altered neuritogenesis 
Human Molecular Genetics  2011;20(12):2344-2355.
The expanded CAG repeat that causes striatal cell vulnerability in Huntington's disease (HD) encodes a polyglutamine tract in full-length huntingtin that is correlated with cellular [ATP] and [ATP/ADP]. Since striatal neurons are vulnerable to energy deficit, we have investigated, in Hdh CAG knock-in mice and striatal cells, the hypothesis that decreased energetics may affect neuronal (N)-cadherin, a candidate energy-sensitive adhesion protein that may contribute to HD striatal cell sensitivity. In vivo, N-cadherin was sensitive to ischemia and to the effects of full-length mutant huntingtin, progressively decreasing in HdhQ111 striatum with age. In cultured striatal cells, N-cadherin was decreased by ATP depletion and STHdhQ111 striatal cells exhibited dramatically decreased N-cadherin, due to decreased Cdh2 mRNA and enhanced N-cadherin turnover, which was partially normalized by adenine supplementation to increase [ATP] and [ATP/ADP]. Consistent with decreased N-cadherin function, STHdhQ111 striatal cells displayed profound deficits in calcium-dependent N-cadherin-mediated cell clustering and cell–substratum adhesion, and primary HdhQ111 striatal neuronal cells exhibited decreased N-cadherin and an abundance of immature neurites, featuring diffuse, rather than clustered, staining for N-cadherin and synaptic vesicle markers, which was partially rescued by adenine treatment. Thus, mutant full-length huntingtin, via energetic deficit, contributes to decreased N-cadherin levels in striatal neurons, with detrimental effects on neurite maturation, strongly suggesting that N-cadherin-mediated signaling merits investigation early in the HD pathogenic disease process.
PMCID: PMC3098733  PMID: 21447599
6.  Genetic variation of aldolase from Korean isolates of Plasmodium vivax and its usefulness in serodiagnosis 
Malaria Journal  2012;11:159.
The malaria aldolase is widely used as rapid diagnostic test (RDT), but the efficacy in aspect of its serological effectiveness in diagnosis is not known. The genetic variation of Korean isolates was analysed and recombinant aldolase was evaluated as a serological antigen in Plasmodium vivax malaria.
Genomic DNA was purified and the aldolase gene of P. vivax from 25 patients’ blood samples was amplified. The samples came from 5 epidemic areas; Bucheon-si, Gimpo-si, Paju-si of Gyeonggido, Gangwha-gun of Incheon metropolitan city, and Cheorwon of Gangwon-do, South Korea. The antigenicity of the recombinant aldolase was tested by western blot and enzyme-linked immunosorbent assay (ELISA).
Sequence analysis of 25 Korean isolates of P. vivax showed that the open reading frame (ORF) of 1,110 nucleotides encoded a deduced protein of 369 amino acids (aa). This ORF showed 100% homology with the P. vivax Sal I strain (XM_00165894) and P. vivax WDK strain (AF247063), 87.4% homology with Plasmodium falciparum (AF179421), 90.6% homology with Plasmodium chabaudi (AF247060), 89.5% homology with Plasmodium vinckei (AF247061), and 96.7% homology with Plasmodium knowlesi. A single nucleotide polymorphism (SNP) at nucleotide 180 (G to A, n = 5) was also observed in the isolates. The expressed recombinant protein had a molecular weight of approximately 31 kDa (monomeric form) and 62 kDa (dimeric form) as analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Among 109 P. vivax patients, 32 (29.4%) had positive in an enzyme-linked absorbance assay (ELISA). This result showed significant correlation between ELISA and an indirect fluorescent antibody test (IFAT) (P < 0.0001).
The aldolase gene from Korean isolates of P. vivax showed one SNP at nucleotide position 180; this SNP mutant was discovered in only the western part of Han River, and included the regions of Ganghwa, Gimpo, and Bucheon. Based on the results, the relationship between antibody production against aldolase and the pattern of disease onset should be more investigated before using aldolase for serodiagnosis.
PMCID: PMC3413559  PMID: 22569198
7.  Fingolimod provides long-term protection in rodent models of cerebral ischemia 
Annals of neurology  2010;69(1):119-129.
The sphingosine-1-phosphate receptor agonist fingolimod (FTY720), that has shown efficacy in advanced multiple sclerosis clinical trials, decreases reperfusion injury in heart, liver and kidney. We therefore tested the therapeutic effects of fingolimod in several rodent models of focal cerebral ischemia. To assess the translational significance of these findings, we asked whether fingolimod improved long-term behavioral outcomes, whether delayed treatment was still effective, and whether neuroprotection can be obtained in a second species.
We used rodent models of middle cerebral artery occlusion and cell culture models of neurotoxicity and inflammation to examine the therapeutic potential and mechanisms of neuroprotection by fingolimod.
In a transient mouse model, fingolimod reduced infarct size, neurological deficit, edema and the number of dying cells in the core and periinfarct area. Neuroprotection was accompanied by decreased inflammation, as fingolimod-treated mice had fewer activated neutrophils, microglia/macrophages, and ICAM-1-positive blood vessels. Fingolimod-treated mice showed a smaller infarct and performed better in behavioral tests up to 15 days after ischemia. Reduced infarct was observed in a permanent model even when mice were treated 4 hours after ischemic onset. Fingolimod also decreased infarct size in a rat model of focal ischemia. Fingolimod did not protect primary neurons against glutamate excitotoxicity or hydrogen peroxide, but decreased ICAM-1 expression in brain endothelial cells stimulated by TNFalpha.
These findings suggest that anti-inflammatory mechanisms, and possibly vasculo-protection, rather than direct effects on neurons, underlie the beneficial effects of fingolimod after stroke. S1P receptors are a highly promising target in stroke treatment.
PMCID: PMC3200194  PMID: 21280082
8.  Comparison of the antibody responses to Plasmodium vivax and Plasmodium falciparum antigens in residents of Mandalay, Myanmar 
Malaria Journal  2011;10:228.
The aim of this study was to investigate the profile of antibodies against several antigens of Plasmodium vivax and Plasmodium falciparum in Mandalay, Myanmar.
Malaria parasites were identified by microscopic examination. To test the antibodies against P. vivax and P. falciparum in sera, an indirect immunofluorescence antibody test (IFAT) was performed using asexual blood parasite antigens. An enzyme-linked immunosorbent assay (ELISA) was performed with circumsporozoite protein (CSP), Pvs25 and Pvs28 recombinant proteins of transmission-blocking vaccine candidates for P. vivax, and liver stage specific antigen-1 and -3 (PfLSA-1, PfLSA-3) for P. falciparum.
Fourteen patients among 112 were found to be infected with P. vivax and 26 with P. falciparum by thick smear examination. Twenty-three patients were found to be infected with P. vivax, 19 with P. falciparum and five with both by thin smear examination. Blood samples were divided into two groups: Group I consisted of patients who were positive for infection by microscopic examination, and Group II consisted of those who showed symptoms, but were negative in microscopic examination. In P. falciparum, IgG against the blood stage antigen in Group I (80.8%) was higher than in Group II (70.0%). In P. vivax, IgG against the blood stage antigen in Group I (53.8%) was higher than in Group II (41.7%). However, the positivity rate of the PvCSP VK210 subtype in Group II (40.0%) was higher than in Group I (23.1%). Similarly for the PvCSP VK247 subtype, Group II (21.7%) was higher than that for Group I (9.6%). A similar pattern was observed in the ELISA using Pvs25 and Pvs28: positive rates of Group II were higher than those for Group I. However, those differences were not shown significant in statistics.
The positive rates for blood stage antigens of P. falciparum were higher in Group I than in Group II, but the positive rates for antigens of other stages (PfLSA-1 and -3) showed opposite results. Similar to P. falciparum, the positive rate of pre-blood stage (CSP VK210 and 247 subtype) and post-blood stage (Pvs25 and 28) antigens of P. vivax were higher in Group II than in Group I. Therefore, sero-diagnosis is not helpful to discriminate between malaria patients and symptomatic individuals during the epidemic season in Myanmar.
PMCID: PMC3163629  PMID: 21819610
9.  Genetic Analysis of the Role of Tumor Necrosis Factor Receptors in Functional Outcome after Traumatic Brain Injury in Mice 
Journal of Neurotrauma  2010;27(6):1037-1046.
We previously reported that tumor necrosis factor-α (TNF-α) and Fas receptor induce acute cellular injury, tissue damage, and motor and cognitive deficits after controlled cortical impact (CCI) in mice (Bermpohl et al. 2007); however, the TNF receptors (TNFR) involved are unknown. Using a CCI model and novel mutant mice deficient in TNFR1/Fas, TNFR2/Fas, or TNFR1/TNFR2/Fas, we tested the hypothesis that the combination of TNFR2/Fas is protective, whereas TNFR1/Fas is detrimental after CCI. Uninjured knockout (KO) mice showed no differences in baseline physiological variables or motor or cognitive function. Following CCI, mice deficient in TNFR2/Fas had worse post-injury motor and Morris water maze (MWM) performance than wild-type (WT) mice (p < 0.05 group effect for wire grip score and MWM performance by repeated measures ANOVA). No differences in motor or cognitive outcome were observed in TNFR1/Fas KO, or in TNFR2 or TNFR1 single KO mice, versus WT mice. Additionally, no differences in propidium iodide (PI)-positive cells (at 6 h) or lesion size (at 14 days) were observed between WT and TNFR1/Fas or TNFR2/Fas KO mice. Somewhat surprisingly, mice deficient in TNFR1/TNFR2/Fas also had PI-positive cells, lesion size, and motor and MWM deficits similar to those of WT mice. These data suggest a protective role for TNFR2/Fas in the pathogenesis of TBI. Further studies are needed to determine whether direct or indirect effects of TNFR1 deletion in TNFR2/Fas KO mice mediate improved functional outcome in TNFR1/TNFR2/Fas KO mice after CCI.
PMCID: PMC2943499  PMID: 20205514
controlled cortical impact; gene knockout; inflammation; mice; tumor necrosis factor; traumatic brain injury
10.  Murine immune responses to a Plasmodium vivax-derived chimeric recombinant protein expressed in Brassica napus 
Malaria Journal  2011;10:106.
To develop a plant-based vaccine against Plasmodium vivax, two P. vivax candidate proteins were chosen. First, the merozoite surface protein-1 (MSP-1), a major asexual blood stage antigen that is currently considered a strong vaccine candidate. Second, the circumsporozoite protein (CSP), a component of sporozoites that contains a B-cell epitope.
A synthetic chimeric recombinant 516 bp gene encoding containing PvMSP-1, a Pro-Gly linker motif, and PvCSP was synthesized; the gene, named MLC, encoded a total of 172 amino acids. The recombinant gene was modified with regard to codon usage to optimize gene expression in Brassica napus. The Ti plasmid inducible gene transfer system was used for MLC chimeric recombinant gene expression in B. napus. Gene expression was confirmed by polymerase chain reaction (PCR), beta-glucuronidase reporter gene (GUS) assay, and Western blot.
The MLC chimeric recombinant protein expressed in B. napus had a molecular weight of approximately 25 kDa. It exhibited a clinical sensitivity of 84.21% (n = 38) and a clinical specificity of 100% (n = 24) as assessed by enzyme-linked immunosorbent assay (ELISA). Oral immunization of BALB/c mice with MLC chimeric recombinant protein successfully induced antigen-specific IgG1 production. Additionally, the Th1-related cytokines IL-12 (p40), TNF, and IFN-γ were significantly increased in the spleens of the BALB/c mice.
The chimeric MLC recombinant protein produced in B. napus has potential as both as an antigen for diagnosis and as a valuable vaccine candidate for oral immunization against vivax malaria.
PMCID: PMC3098821  PMID: 21529346
11.  Blocking Effect of a Monoclonal Antibody against Recombinant Pvs25 on Sporozoite Development in Anopheles sinensis▿  
To develop a vaccine to block the transmission of vivax malaria, the gene encoding the ookinete surface protein Pvs25 was cloned from a Korean malaria patient. The Pvs25 gene was 660 bp long, encoding 219 amino acids. It was subcloned into the expression vector pQE30 and expressed in Escherichia coli. The expressed recombinant protein, named rPvs25, showed a molecular mass of approximately 25 kDa by SDS-PAGE analysis. An anti-rPvs25 monoclonal antibody produced in BALB/c mice was able to inhibit sporozoite development in the mosquito Anopheles sinensis, which is known as the malaria transmission vector in the Republic of Korea. In addition, rPvs25 produced a relatively high antibody titer in BALB/c mice that lasted for more than 6 months. Based on these results, we suggest that recombinant Pvs25 could be a useful antigen in the development of a vaccine to prevent local malaria transmission in the Republic of Korea.
PMCID: PMC2916255  PMID: 20554802
12.  Detection of an antibody against Plasmodium vivax in residents of Gimpo-si, South Korea, using an indirect fluorescent antibody test 
Malaria Journal  2011;10:19.
First reemerged malaria case was reported in 1993 after two decades absent in South Korea. Thereafter, Plasmodium vivax spreads out near demilitarized zone (DMZ). This study investigated the prevalence of P. vivax after the malaria transmission season in Gimpo-si where adjacent to DMZ of South Korea. An indirect fluorescent antibody test (IFAT) was performed to evaluate anti-malaria antibodies in blood samples.
Microscopic examinations were performed to identify the presence of malaria parasites. Antibodies against P. vivax were detected using IFAT, and blood samples from antibody-positive cases were tested using a polymerase chain reaction (PCR) assay that detects malaria parasites.
A total of 5,797 blood samples were collected from residents in Gimpo-si. The positivity rate by IFAT was 2.16% (n = 125). Yangchon-myeon (3.28%) had the highest positivity rate of the seven administrative districts tested. Positivity rates increased with age (P < 0.05). Sixteen of the IFAT positive samples (12.80%, n = 125) were positive for malaria DNA according to PCR. Blood samples with an antibody titer over 1:256 had high positivity rates in the PCR analysis (P < 0.05).
These results indicate that antibody titers obtained using IFAT may provide useful information about the prevalence of P. vivax in low endemic areas and could be used to detect asymptomatic patients. Finding asymptomatic patients is important in eliminating vivax malaria in South Korea.
PMCID: PMC3042984  PMID: 21281481
13.  Analysis of the dihydrofolate reductase-thymidylate synthase gene sequences in Plasmodium vivax field isolates that failed chloroquine treatment 
Malaria Journal  2010;9:331.
To use pyrimethamine as an alternative anti-malarial drug for chloroquine-resistant malaria parasites, it was necessary to determine the enzyme's genetic variation in dihydrofolate reductase-thymidylate syntase (DHFR-TS) among Korean strains.
Genetic variation of dhfr-ts genes of Plasmodium vivax clinical isolates from patients who did not respond to drug treatment (n = 11) in Korea were analysed. The genes were amplified using the polymerase chain reaction (PCR) with genomic DNA as a template.
Sequence analysis showed that the open reading frame (ORF) of 1,857 nucleotides encoded a deduced protein of 618 amino acids (aa). Alignment with the DHFR-TS genes of other malaria parasites showed that a 231-residue DHFR domain and a 286-residue TS domain were seperated by a 101-aa linker region. This ORF shows 98.7% homology with the P. vivax Sal I strain (XM001615032) in the DHFR domain, 100% in the linker region and 99% in the TS domain. Comparison of the DHFR sequences from pyrimethamine-sensitive and pyrimethamine-resistant P. vivax isolates revealed that nine isolates belonged to the sensitive strain, whereas two isolates met the criteria for resistance. In these two isolates, the amino acid at position 117 is changed from serine to asparagine (S117N). Additionally, all Korean isolates showed a deletion mutant of THGGDN in short tandem repetitive sequences between 88 and 106 amino acid.
These results suggest that sequence variations in the DHFR-TS represent the prevalence of antifolate-resistant P. vivax in Korea. Two of 11 isolates have the Ser to Asn mutation in codon 117, which is the major determinant of pyrimethamine resistance in P. vivax. Therefore, the introduction of pyrimethamine for the treatment of chloroquine-resistant vivax malaria as alternative drug in Korea should be seriously considered.
PMCID: PMC2999615  PMID: 21087471
14.  Prevalence of Plasmodium vivax VK210 and VK247 subtype in Myanmar 
Malaria Journal  2010;9:195.
Plasmodium vivax is divided into two subtypes, a dominant form, VK210 and a variant form, VK247. This division is dependent on the amino acid composition of the circumsporozoite (CS) protein. In this study, the prevalence of the VK247 variant form of P. vivax was investigated in Myanmar.
The existence of malaria parasites in blood samples was determined by microscopic examination, polymerase chain reaction (PCR) and DNA hybridization assays. To test for antibodies against P. vivax and Plasmodium falciparum in blood samples, an indirect immunofluorescence antibody test (IFAT) was performed using asexual blood antigens. An enzyme-linked immunosorbent assay with synthetic VK210 and VK247 antigens was carried out to discriminate between the P. vivax subtypes.
By thick smear examination, 73 (n = 100) patients were single infected with P. vivax, one with P. falciparum and 13 with both species. By thin smear, 53 patients were single infected with P. vivax, eight with only P. falciparum and 16 with both. Most of the collected blood samples were shown to be P. vivax positive (n = 95) by PCR. All cases that were positive for P. falciparum by PCR (n = 43) were also positive for P. vivax. However, 52 cases were single infected with P. vivax. IFAT showed antibody titres from 1:32 to 1:4,096. Additionally, using specific antibodies for VK210 and VK247, ELISA showed that 12 patients had antibodies for only the VK210 subtype, 4 patients had only VK247 subtype antibodies and 21 patients had antibodies for both subtypes. Using a DNA hybridization test, 47 patients were infected with the VK210 type, one patient was infected with VK247 and 23 patients were infected with both subtypes.
The proportion of the VK247 subtype in Myanmar was 43.1% (n = 25) among 58 positive cases by serodiagnosis and 25.6% (n = 24) among 94 positive cases by genetic diagnosis. In both diagnostic methods, the infection status of malaria patients is highly diverse with respect to malaria species, and multiple clonal infections are prevalent in Myanmar. Therefore, the complexity of the infection should be considered carefully when diagnosing malaria in Myanmar.
PMCID: PMC2914060  PMID: 20615261
15.  Rac1 Is a Critical Mediator of Endothelium-Derived Neurotrophic Activity 
Science signaling  2009;2(61):ra10.
The therapeutic potential of neurotrophic factors has been hampered by their inability to achieve adequate tissue penetration. Brain blood vessels, however, could be an alternative target for neuro-salvage therapies by virtue of their close proximity to neurons. Here we show that hemizygous deletion of Rac1 in mouse endothelial cells (ECs) attenuates brain injury and edema after focal cerebral ischemia. Microarray analysis of Rac1+/− ECs revealed enrichment of stress response genes, basement membrane components, and neurotrophic factors that could affect neuronal survival. Consistent with these expression profiles, endothelial Rac1 hemizygosity enhanced antioxidative and endothelial barrier capacities and potentiated paracrine neuroprotective activities through the up-regulation of the neurotrophic factor, artemin. Endothelial Rac1, therefore, could be an important therapeutic target for promoting endothelial barrier integrity and neurotrophic activity.
PMCID: PMC2668716  PMID: 19278959
16.  Obesity Increases Vascular Senescence and Susceptibility to Ischemic Injury Through Chronic Activation of Akt and mTOR 
Science signaling  2009;2(62):ra11.
Obesity and age are important risk factors for cardiovascular disease. However, the signaling mechanism linking obesity with age-related vascular senescence is unknown. Here we show that mice fed a high-fat diet show increased vascular senescence and vascular dysfunction compared to mice fed standard chow and are more prone to peripheral and cerebral ischemia. All of these changes involve long-term activation of the protein kinase Akt. In contrast, mice with diet-induced obesity that lack Akt1 are resistant to vascular senescence. Rapamycin treatment of diet-induced obese mice or of transgenic mice with long-term activation of endothelial Akt inhibits activation of mammalian target of rapamycin (mTOR)–rictor complex 2 and Akt, prevents vascular senescence without altering body weight, and reduces the severity of limb necrosis and ischemic stroke. These findings indicate that long-term activation of Akt-mTOR signaling links diet-induced obesity with vascular senescence and cardiovascular disease.
PMCID: PMC2667954  PMID: 19293429
17.  Additive effects of statin and dipyridamole on cerebral blood flow and stroke protection 
Recent studies suggest that dipyridamole (DP) may exert stroke protective effects beyond platelet inhibition. The purpose of this study is to determine whether statin and DP could enhance stroke protection through nitric oxide (NO)-dependent vascular effects. Mice were pretreated with DP (10 to 60 mg/kg, q 12 h, 3 days) alone or in combination with a statin (simvastatin; 0.1 to 20 mg/kg per day, 14 days) before transient intraluminal middle cerebral artery occlusion. Although simvastatin (1 mg/kg per day, 14 days) increased endothelial NO synthase (eNOS) activity by 25% and DP (30 mg/kg, q12 h, 3 days) increased aortic cGMP levels by 55%, neither statin nor DP alone, at these subtherapeutic doses, increased absolute cerebral blood flow (CBF) or conferred stroke protection. However, the combination of subtherapeutic doses of simvastatin and DP increased CBF by 50%, decreased stroke volume by 54%, and improved neurologic motor deficits, all of which were absent in eNOS-deficient mice. In contrast, treatment with aspirin (10 mg/kg per day, 3 days) did not augment the neuroprotective effects of DP and/or simvastatin. These findings indicate that statin and DP exert additive NO-dependent vascular effects and suggest that the combination of statin and DP has greater benefits in stroke protection than statin alone through vascular protection.
PMCID: PMC2662038  PMID: 18382469
aspirin; blood flow; cerebral ischemia; endothelium; nitric oxide synthase; stroke
18.  Decreased Perivascular Fibrosis but Not Cardiac Hypertrophy in ROCK1+/− Haploinsufficient Mice 
Circulation  2005;112(19):2959-2965.
Rho GTPase and its downstream target, Rho-associated kinase (ROCK), have been implicated in diverse cardiovascular diseases such as cardiac hypertrophy. However, pharmacological inhibitors of ROCK are not entirely specific, nor can they discriminate between the ROCK isoforms ROCK1 and ROCK2. To determine the specific role of ROCK1 in the development of cardiac hypertrophy, we generated ROCK1+/− haploinsufficient mice and determined whether cardiac hypertrophy and remodeling are decreased in these mice.
Methods and Results
Litters of ROCK1−/− mice on C57Bl/6 background were markedly underrepresented, suggesting lethality in utero or postnatally. ROCK1+/− mice, however, are viable and fertile with no obvious phenotypic abnormalities. Basal blood pressure, heart rate, and cardiac dimension and function in ROCK1+/− mice were similar to those in wild-type (WT) littermates. Infusion of angiotensin II (400 ng · kg−1 · min−1 for 28 days) or treatment with NG-nitro-l-arginine methyl ester (1 mg/mL in drinking water for 28 days) caused similar increases in systolic blood pressure, left ventricular wall thickness, left ventricular mass, ratio of heart weight to tibial length, and cardiomyocyte size in ROCK1+/− mice and WT littermates. In contrast, perivascular fibrosis in hearts was increased to a lesser extent in ROCK1+/− mice compared with WT littermates. This was associated with decreased expression of transforming growth factor-β, connective tissue growth factor, and type III collagen. In addition, perivascular fibrosis induced by transaortic constriction or myocardial infarction was decreased in ROCK1+/− mice compared with WT littermates.
These findings indicate ROCK1 is critical for the development of cardiac fibrosis, but not hypertrophy, in response to various pathological conditions and suggest that signaling pathways leading to the hypertrophic and profibrotic response of the heart are distinct.
PMCID: PMC2640100  PMID: 16260635
blood pressure; hypertension; hypertrophy; remodeling; angiotensin
19.  Inhibition of Rho Kinase (ROCK) Leads to Increased Cerebral Blood Flow and Stroke Protection 
Background and Purpose
Endothelium-derived nitric oxide (NO) plays a pivotal role in vascular protection. The Rho kinase (ROCK) inhibitor, hydroxyfasudil, prevents the downregulation of endothelial NO synthase (eNOS) under hypoxic conditions. However, it is unknown whether inhibition of ROCK can attenuate ischemia-induced endothelial dysfunction and tissue damage in vivo.
Human vascular endothelial cells were treated with increasing concentrations of hydroxyfasudil (0.1 to 100 μmol/L) and eNOS expression and activity were measured. To determine the physiological relevance of eNOS regulation by ROCK, we administered fasudil, which is metabolized to hydroxyfasudil in vivo, to mice for 2 days before subjecting them to middle cerebral artery occlusion. Cerebral blood flow, cerebral infarct size, and neurologic deficit were measured.
In a concentration-dependent manner, hydroxyfasudil increased eNOS mRNA and protein expression, resulting in a 1.9- and 1.6-fold increase, respectively, at 10 μmol/L (P<0.05 for both). This correlated with a 1.5- and 2.3-fold increase in eNOS activity and NO production, respectively (P<0.05 for both). Fasudil increased cerebral blood flow to both ischemic and nonischemic brain areas, reduced cerebral infarct size by 33%, and improved neurologic deficit score by 37% (P<0.05). This correlated with inhibition of brain and vascular ROCK activity and increased eNOS expression and activity. Another ROCK inhibitor, Y-27632, also showed similar effects. The neuroprotective effects of fasudil were absent in eNOS-deficient mice.
These findings indicate that the neuroprotective effect of ROCK inhibition is mediated by endothelium-derived NO and suggest that ROCK may be an important therapeutic target for ischemic stroke.
PMCID: PMC2633591  PMID: 16141422
cerebral blood flow; nitric oxide; stroke
20.  Translational Therapeutics of Dipyridamole 
Dipyridamole (DP) is a phosphodiesterase inhibitor that increases the intracellular levels of cyclic adenosine monophosphate (cAMP) and cyclic guanine monophosphate (cGMP) by preventing their conversion to AMP and GMP, respectively. By increasing cAMP and cGMP levels in platelets, DP reversibly inhibits platelet aggregation and platelet-mediated thrombotic disease. In addition, DP may potentiate some of the vascular protective effects of endothelium-derived nitric oxide (NO), which increases cGMP by stimulating soluble guanylyl cyclase. Endothelium-derived NO is an important regulator of vascular tone, blood flow, and tissue perfusion. Indeed, endothelial NO synthase-deficient (eNOS−/−) mice exhibit elevated systemic blood pressure and have larger myocardial and cerebral infarct size after ischemic injury. Other NO/cGMP-dependent effects that may be potentiated by DP include inhibition of vascular smooth muscle proliferation and prevention of endothelial-leukocyte interaction. In addition, DP increases local concentrations of adenosine and prostacyclin, which could affect vascular tone and inflammation. Finally, DP has antioxidant properties, which could stabilize platelet and vascular membranes as well as prevent the oxidation of low-density lipoprotein. These platelet and nonplatelet actions of DP may contribute to some of its therapeutic benefits in vascular disease.
PMCID: PMC2615560  PMID: 18174451
platelets; endothelium; vascular; dipyridamole; oxidation; inflammation; perfusion
21.  Regulation of Endothelial Nitric Oxide Synthase and Postnatal Angiogenesis by Rac1 
Circulation research  2008;103(4):360-368.
Diminished bioavailability of nitric oxide is a hallmark of endothelial dysfunction and is associated with a broad spectrum of vascular disorders such as impaired angiogenesis. Because Rac1, a Rho family member, mediates cellular motility and generation of reactive oxygen species, it could be involved in the regulation of endothelial nitric oxide production. However, the pathophysiological consequences of postnatal endothelial Rac1 deletion on endothelial function have not been determined. We generated endothelial-specific Rac1 haploinsufficient mice (EC-Rac1+/-) using Cre-loxP technology. The EC-Rac1+/- mice have decreased expression and activity of endothelial nitric oxide synthase (eNOS), impaired endothelium-dependent vasorelaxation, and mild hypertension compared with control (Rac1+/flox) mice. Hind limb ischemia model and aortic capillary sprouting assay showed that eNOS activity and angiogenesis was impaired in EC-Rac1+/- mice. Indeed, Rac1 promotes eNOS gene transcription through p21-activated kinase but not NADPH oxidase, increases eNOS mRNA stability, and enhances eNOS activity by promoting endothelial uptake of l-arginine. These findings indicate that endothelial Rac1 is essential for endothelium-dependent vasomotor response and ischemia-induced angiogenesis. These effects of Rac1 on endothelial function are largely due to the upregulation of eNOS through multiple mechanisms that are mediated, in part, by p21-activated kinase. Therapeutic strategies to enhance Rac1 function, therefore, may be important for preventing endothelial dysfunction.
PMCID: PMC2615563  PMID: 18599867
angiogenesis; endothelium; hypertension; nitric oxide synthase; signal transduction
22.  Decreased vascular lesion formation in mice with inducible endothelial-specific expression of protein kinase Akt 
Journal of Clinical Investigation  2006;116(2):334-343.
To determine whether endothelial Akt could affect vascular lesion formation, mutant mice with a constitutively active Akt transgene, which could be inducibly targeted to the vascular endothelium using the tet-off system (EC-Akt Tg mice), were generated. After withdrawal of doxycycline, EC-Akt Tg mice demonstrated increased endothelial-specific Akt activity and NO production. After blood flow cessation caused by carotid artery ligation, neointimal formation was attenuated in induced EC-Akt Tg mice compared with noninduced EC-Akt Tg mice and control littermates. To determine the role of eNOS in mediating these effects, mice were treated with Nω-nitro-L-arginine methyl ester (L-NAME). Neointimal formation was attenuated to a lesser extent in induced EC-Akt Tg mice treated with L-NAME, suggesting that some of the vascular protective effects were NO independent. Indeed, endothelial activation of Akt resulted in less EC apoptosis in ligated arteries. Immunostaining demonstrated decreased inflammatory and proliferative changes in induced EC-Akt Tg mice after vascular injury. These findings indicate that endothelial activation of Akt suppresses lesion formation via increased NO production, preservation of functional endothelial layer, and suppression of inflammatory and proliferative changes in the vascular wall. These results suggest that enhancing endothelial Akt activity alone could have therapeutic benefits after vascular injury.
PMCID: PMC1359051  PMID: 16453020

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