Biomedical subjects

Guo-Yuan Yang

Publications and source records attributed to Guo-Yuan Yang.

30 records · Page 2Linked to original sources

PubMed2004

Doxycycline suppresses cerebral matrix metalloproteinase-9 and angiogenesis induced by focal hyperstimulation of vascular endothelial growth factor in a mouse model.

BACKGROUND AND PURPOSE: A number of central nervous system (CNS) disorders are associated with abnormalities in or activation of angiogenesis, including vascular malformations. To test the hypothesis that the nonspecific matrix metalloproteinase (MMP) inhibitor, doxycycline, suppresses vascular endothelial growth factor (VEGF)-induced cerebral angiogenesis through inhibition of MMPs, we used a mouse model with enhanced cerebral angiogenesis induced by focal hyperstimulation of VEGF from adenovirus DNA (AdVEGF) transduction. METHODS: The time course study of MMP activity was performed at 7 and 14 days after AdVEGF transduction. MMP activity and expression were examined by zymography and immunohistochemistry, respectively. As an index of cerebral angiogenesis, microvessel counting was performed in the brains of 3 groups of mice (n=6): (1) control; (2) AdVEGF only; and (3) AdVEGF plus doxycycline (30 mg/kg per day). RESULTS: Brain MMP-9 activities increased 4-fold (883+/-137 versus 179+/-179; 1-sided P<0.001) at 7 days after AdVEGF transduction. VEGF transduction increased vessel counts by 19% (255+/-27 versus 215+/-15, 1-sided P<0.01). Doxycycline treatment decreased MMP-9 activity (89+/-72 versus 883+/-137; 1-sided P<0.001) and cerebral microvessel number (231+/-17 versus 255+/-27; 1-sided P<0.05). CONCLUSIONS: Doxycycline is effective in decreasing stimulated cerebral MMP-9 activity and parenchymal angiogenesis. The decrease in MMP-9 activity is associated with decreased microvessel counts. Brain pathophysiological processes that involve abnormally enhanced angiogenesis may be amenable to manipulation by MMP inhibitors, including tetracycline derivatives.

Adenoviridae↗

PubMed2004

PI3K inhibition in neonatal rat brain slices during and after hypoxia reduces phospho-Akt and increases cytosolic cytochrome c and apoptosis.

Acute hypoxia can deplete ATP and induce mitochondrial release of cytochrome c (cyt c) to initiate or enhance apoptosis, a process delayed or overcome with sufficient ATP and phosphorylation (activation) of survival factors such as Akt (also known as Protein Kinase B). We used an ex vivo brain slice model to investigate associations between levels of phosphorylated Akt (phospho-Akt) and the extent of intrinsic pathway apoptosis. Additionally, phosphorylation (inactivation) was measured of Bad, which is known to promote mitochondrial release of cyt c. Superfused cerebrocortical slices from 7-day-old rats underwent 30-min hypoxia followed by 4-h reoxygenation. At end-hypoxia, Western blots of phospho-Akt became nearly undetectable but returned immediately during recovery and increased thereafter. Cyt c behaved oppositely, being greatest at end-hypoxia and continually decreasing during recovery. Continuous inhibition of phosphoinositide 3-kinase (PI3K) with 10 microM LY294002 suppressed post-hypoxic phospho-Akt levels, prevented post-hypoxic cytosolic cyt c reductions, and increased apoptosis evaluated by TUNEL staining and DNA fragmentation. Western blot analysis demonstrated enhanced Bad translocation from cytosol to mitochondria in the LY294002 group. Phospho-Akt/phospho-Bad double staining revealed colocalization. Parallel (31)P NMR studies showed no effects on NTP production by LY294002. The data support prominent roles for Bad phosphorylation in phospho-Akt's reduction of cyt c apoptosis, and possible apoptotic roles at mitochondrial targets of Bad.

Animals↗

PubMed2004

Inhibition of MEK/ERK 1/2 pathway reduces pro-inflammatory cytokine interleukin-1 expression in focal cerebral ischemia.

It has been proposed that mitogen-activated protein kinase (MAPK) pathways may play a role in the regulation of pro-inflammatory cytokines, such as interlukine-1, during cerebral ischemia. Our previous study showed that extracellular-signal-regulated kinases 1 and 2 (ERK 1/2) were activated during focal cerebral ischemia in mice [J. Cereb. Blood Flow Metab. 20 (2000) 1320]. However, the effect of ERK 1/2 activation in focal cerebral ischemia is still unclear. In this study we reported that in vivo phospho-ERK 1/2 expression increased following 30 min of middle cerebral artery occlusion (MCAO) in the mouse brain in both the ischemic core and perifocal regions. Western blot analysis and immunohistochemistry demonstrated that pro-treatment with 1,4-diamino-2,3-dicyano-1,4-bis butadiene (U0126) [J. Biol. Chem. 273 (1998) 18623] could significantly inhibit mouse brain phospho-MEK 1/2 and phospho-ERK 1/2 expression after 1-2 h of MCAO (p<0.05). Compared to the control group of mice, brain infarct volume was significantly decreased after 24 h of MCAO in the U0126-treated mice (27+/-6 vs. 46+/-9 mm(2), p<0.05). Inhibition of the MEK/ERK 1/2 pathway also prevented downstream kinase Elk-1 phosphorylation, and further reduced cytokine IL-1beta mRNA, but not TNFalpha, IL-1alpha, or chemokine MIP-1alpha mRNA expression. Our data demonstrates that in vivo the close linking of MEK 1/2, ERK 1/2, Elk-1, and IL-1 mRNA expression in the cerebral ischemia animals suggests that ERK 1/2 pathway activation is important in pro-inflammatory cytokine IL-1beta signaling, which induces an inflammatory response and exacerbates ischemic brain injury. Inhibiting the ERK 1/2 pathway may therefore provide a novel approach for the reduction of ischemia-induced IL-1beta overexpression.

Animals↗

PubMed2004

Using 31P NMR spectroscopy at 14.1 Tesla to investigate PARP-1 associated energy failure and metabolic rescue in cerebrocortical slices.

PARP-1 activation by H(2)O(2) in an acute preparation of superfused, respiring, neonatal cerebrocortical slices was assessed from PAR-polymer formation detected with immunohistochemistry and Western blotting. (31)P NMR spectroscopy at 14.1 Tesla of perchloric acid slice extracts was used to assess energy failure in a 1-h H(2)O(2) exposure as well as in a subsequent 4-h recovery period where the superfusate had no H(2)O(2) and specifically chosen metabolic substrates. Although more data are needed to fully characterize different bioenergetic responses, a high NMR spectral resolution (PCr full-width at half-max approximately.01 ppm) and narrow widths for most metabolites (<.2 ppm) permitted accurate quantifications of spectrally resolved resonances for ADP, ATP, NAD(+)/NADH, and other high energy phosphates. It appears possible to use brain slices to quantitatively study PARP-related, NAD-associated energy failure, and rescue with TCA metabolites.

Animals↗

PubMed2004

Vascular endothelial growth factor induces abnormal microvasculature in the endoglin heterozygous mouse brain.

Hereditary hemorrhagic telangiectasia (HHT), associated with brain arteriovenous malformations, is caused by a loss of function mutation in either the endoglin (HHT1) or activin receptor-like kinase 1 gene (ALK-1, HHT2). Endoglin heterozygous (Eng+/-)mice have been proposed as a disease model. To better understand the role of endoglin in vascular malformation development, we examined the effect of vascular endothelial growth factor (VEGF) hyperstimulation on microvessels in adult endoglin heterozygous (Eng+/-) mice using an adenoviral vector to deliver recombinant human VEGF165 cDNA (AdhVEGF) into basal ganglia. VEGF expression was increased in AdhVEGF mice compared with the AdlacZ and saline group (P < 0.05) and localized to multiple cell types (neurons, astrocytes, endothelial cells, and smooth muscle cells) by double-labeled immunostaining. VEGF overexpression increased microvessel count for up to 4 weeks in both the Eng+/+ and Eng+/- groups (Eng+/+ 185 +/- 14 vs. Eng+/- 201 +/- 10 microvessels/mm2). Confocal microscopic examination revealed grossly abnormal microvessels in eight of nine Eng+/- mouse brains compared with zero of nine in Eng+/+ mice (P < 0.05). Abnormal microvessels featured enlargement, clustering, twist, or spirals. VEGF receptor Flk-1 and TGF-beta receptor 1 (T beta R1) expression were reduced in the Eng+/- mouse brain compared with control. Excessive VEGF stimulation may play a pivotal role in the initiation and development of brain vessel malformations in states of relative endoglin insufficiency in adulthood. These observations are relevant to our general understanding of the maintenance of vascular integrity.

Animals↗

PubMed2004

Retroviral delivery of homeobox D3 gene induces cerebral angiogenesis in mice.

Angiogenesis is regulated by concerted actions of angiogenic and angiostatic factors. Homeobox D3 gene (HOXD3) is a potent proangiogenic transcription factor that promotes angiogenesis by modulating the expression of matrix-degrading proteinases, integrins, and extracellular matrix components. Application of HOXD3 can promote angiogenesis in the skin, but its role in other vascular beds has not been examined. The authors examined HOXD3 expression in human brain vessels by in situ hybridization. Although little or no HOXD3 mRNA was detected in normal brain vessels, increased levels of HOXD3 and its target gene, alpha V beta 3, were found in angiogenic vessels in human brain arteriovenous malformations. The authors further investigated whether HOXD3 plays a role in cerebral angiogenesis in a murine model. Expression of HOXD3 in mouse brain was achieved through retroviral vector-mediated HOXD3 gene transfer. HOXD3 expression lead to a significant induction of cerebral angiogenesis as shown by quantitative microvessel counting (HOXD3: 241 +/- 19 vessels/mm2 vs. saline: 150 +/- 14 vessels/mm2, P < 0.05). The data also showed that focal cerebral blood flow was increased in the angiogenic region with less vascular leakage. Moreover, expression of HOXD3 led to an increase in the expression of a direct downstream target gene alpha V beta 3 integrin. The data suggest that HOXD3 may play an important role in regulating cerebral angiogenesis, and that gene transfer of HOXD3 may provide a novel and potent means to stimulate angiogenesis.

Animals↗

PubMed2004

Gene microarray analysis of human brain arteriovenous malformations.

OBJECTIVE: Human brain arteriovenous malformations (BAVMs) display abnormal expression of various angiogenesis-related genes and their products. We examined gene expression patterns in BAVMs by the gene microarray technique. METHODS: We analyzed BAVM and control brain samples obtained by temporal lobectomy for medically intractable seizure by Affymetrix Human Gene Set U95Av2 (Affymetrix, Inc., Santa Clara, CA). The gene microarray data were compared with new and previously published data that used conventional molecular biology techniques. RESULTS: We analyzed six BAVM and five control brain samples. From 12,625 gene probes assayed, 1781 gene probes showed differential expression between BAVMs and controls. BAVM samples had a gene expression pattern that was distinct from those of control brain samples. Increased messenger ribonucleic acid expression of vascular endothelial growth factor A was accompanied by increased expression of its protein product. A majority of the gene data was in agreement with previously published data. The gene microarray data generated a new testable hypothesis regarding integrin, and we found increased expression of integrin alphavbeta3 protein in BAVMs. CONCLUSION: The gene expression pattern of BAVMs was distinct from those of control brain samples. We verified the gene microarray data by demonstrating that increased gene expression levels for angiogenesis-related molecules were accompanied by increased levels of their protein product expression. The gene microarray technique may be a useful tool to study multiple pathways simultaneously in BAVM specimens.

Adolescent↗

PubMed2004

Inflammatory reaction after focal cerebral ischemia in mouse.

BACKGROUND: In response to the inflammatory reaction, circulating leukocytes aggregate and adhere to the endothelial cells and eventually pervade into tissues, resulting in cell damage. This study was to detect the inflammatory reactions in mouse focal cerebral ischemia and their distinct characteristics in the ischemic basal ganglia and surrounding cortex. METHODS: Mice were subjected to permanent occlusion of the left middle cerebral artery (MCAO) by introducing a suture for 2 to 120 hours. The expression of intercellular adhesion molecule 1 (ICAM-1) and Mac-1 was determined immunohistochemically. The myeloperoxidase (MPO) activity of the ischemic regions was measured. RESULTS: Four hours after MCAO, the number of ICAM-1 positive vessels in the ischemic basal ganglia increased (9.2 +/- 2.8 per mm(2)), peaked at 48 hours (29.6 +/- 4.8 per mm(2)), and decreased after 72 hours. In the ischemic cortex, the number increased rapidly 4 hours after MCAO (19.4 +/- 6.1 per mm(2)), peaked at 48 hours (44.4 +/- 16.8 per mm(2)), and declined after 72 hours. Mac-1 positive cells were seen in the ischemic basal ganglia (3.4 +/- 1.2 per mm(2)) 12 hours after MCAO, peaked after 48 hours (20.2 +/- 6.3 per mm(2)), and decreased after 72 hours. In the ischemic cortex, however, the number increased 4 hours after MCAO (4.3 +/- 1.7 per mm(2)), peaked after 48 hours (20.9 +/- 8.4 per mm(2)), and remained high at 120 hours. The MPO activity increased in the ischemic basal ganglia 12 hours after MCAO (0.111 +/- 0.023 U/g), peaked after 24 hours (0.194 +/- 0.059 U/g), and decreased after 72 hours. In the ischemic cortex, the MPO activity increased 12 hours after MCAO (0.110 +/- 0.032 U/g), peaked after 24 hours (0.210 +/- 0.067 U/g), and remained elevated at 120 hours. CONCLUSIONS: The increased expression of ICAM-1 in the ischemic brain of mouse in the early phase of MCAO followed by the over-expression of Mac-1 and the increased MPO activity suggests that focal ischemia leads to early onset of inflammation. The inflammatory response is more persistent and intensive in the ischemic cortex than in the ischemic basal ganglia.

Animals↗

PubMed2003

Early NFkappaB activation is inhibited during focal cerebral ischemia in interleukin-1beta-converting enzyme deficient mice.

Our previous study demonstrated that the inhibition of interleukin-1beta (IL-1beta) reduces ischemic brain injury; however, the molecular mechanism of the action of IL-1 in cerebral ischemia is unclear. We are investigating currently the role of NFkappaB during focal cerebral ischemia, using mutant mice deficient in the interleukin-1 converting enzyme gene (ICE KO) in a middle cerebral artery occlusion (MCAO) model. Adult male ICE KO and wild-type mice (n = 120) underwent up to 24 hr of permanent MCAO. Cytoplasmic phospho-NFkappaB/p65 expression in ischemic brain was examined using Western blot analysis and immunohistochemistry. NFkappaB DNA-binding activity was detected using electrophoretic mobility shift assay (EMSA). Furthermore, ICAM-1 expression was examined in both the ICE KO and wild-type mice (WT). Western blot analysis and immunostaining showed that the level of cytosolic phosphorylated NFkappaB/p65 increased after 2 and 4 hr of MCAO in WT mice; however, NFkappaB/p65 was significantly reduced after MCAO in the ICE KO mice (P < 0.05). EMSA showed that NFkappaB DNA-binding activity increased after MCAO in WT mice; but this effect was reduced in the ICE KO mice. The number of ICAM-1-positive vessels in the ischemic hemisphere was greatly attenuated in the ICE KO mice (P < 0.05), which paralleled the results of immunohistochemistry. Our results demonstrate that NFkappaB phosphorylation is reduced in ICE KO mice, suggesting that ICE or IL-1 are involved in early NFkappaB phosphorylation. Because cerebral ischemia induced infarction is significantly reduced in ICE KO mice, we conclude that early NFkappaB phosphorylation plays a disruptive role in the ischemic process.

Animals↗

PubMed2003

Abnormal expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in brain arteriovenous malformations.

BACKGROUND AND PURPOSE: Excessive degradation of the vascular matrix by matrix metalloproteinases (MMPs) can lead to structural instability of vessels. In this study we examined the expression of MMPs and tissue inhibitors of metalloproteinases (TIMPs) in brain arteriovenous malformations (BAVMs). METHODS: We performed gelatin zymography for MMPs and Western blot for MMP-9, MMP-2, TIMP-1, TIMP-2, TIMP-3, and TIMP-4. MMP-9 expression was localized by immunohistochemistry. RESULTS: We analyzed 37 BAVM specimens and 9 control brain specimens from epilepsy surgery. Thirty-two BAVM patients had embolization treatment before resection. Eighteen BAVM patients had a history of hemorrhage from BAVMs. Neither MMP-2 nor TIMP-2 was detected in BAVMs or control brain specimens. Compared with control brain samples, BAVM samples had higher levels of total MMP-9, active MMP-9, pro-MMP-9, TIMP-1, and TIMP-3. TIMP-4 levels were higher in the control brain than in BAVM specimens. MMP-9 was localized to the endothelial cell/peri-endothelial cell layer and infiltrating neutrophils of BAVMs. BAVMs with venous stenosis >or=50% had higher expression of MMP-9 than BAVMs with venous stenosis <50%. There was no apparent association between total MMP-9, pro-MMP-9, or active MMP-9 levels and (1) feeding artery pressure, (2) pattern of draining vein (exclusively deep venous drainage versus any superficial drainage), and (3) BAVM size. CONCLUSIONS: We found increased levels of MMP-9 and TIMPs in BAVMs. Abnormal balance of MMP-9 and TIMPs may contribute to vascular instability of BAVMs.

Adult↗

PubMed2003

Induction of focal angiogenesis through adenoviral vector mediated vascular endothelial cell growth factor gene transfer in the mature mouse brain.

Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen and morphogen, which stimulates angiogenesis in a wide variety of tissues and lesions in vivo. In this study, we applied adenoviral vector delivered human VEGF165 cDNA to develop focal non-tumor angiogenesis in the mature mouse brain. Seventy-two adult CD-1 mice underwent Ad h VEGF, Ad lacZ, and saline injection for up to fourweeks. An adenoviral suspension containing 1 x 10(9) particles was injected stereotactically into the right hemisphere of the brain. The results showed that VEGF expression was increased in the Ad h VEGF transduced mice compared to Ad lacZ or saline injected mice ( P < 0.05). VEGF-positive cells were mainly located in the injection hemisphere of Ad h VEGF transduced mice. Quantitative vessel counting showed that microvessels in the Ad h VEGF transduced mice increased following 2 weeks of Ad h VEGF gene transfer compared to the other two groups (Ad h VEGF:241 +/- 19 vs. Ad lacZ :148 +/- 17 and Saline:150 +/- 14 vessels/mm2, P < 0.05). Morphology showed typical angiogenic changes. PCNA-positive staining confirmed these microvessels were actively proliferating. Our study demonstrates that Ad h VEGF-induced VEGF hyper-stimulation causes focal angiogenesis in the mature mouse brain. This novel method of inducing in vivo brain focal angiogenesis provides an opportunity to study the molecular mechanisms independent of the confounding effects of upstream inciting stimuli such as ischemia or tumor.

Adenoviridae↗

PubMed2002

Transforming growth factor-beta 1 increases bad phosphorylation and protects neurons against damage.

Despite the characterization of neuroprotection by transforming growth factor-beta1 (TGF-beta1), the signaling pathway mediating its protective effect is unclear. Bad is a proapoptotic member of the Bcl-2 family and is inactivated on phosphorylation via mitogen-activated protein kinase (MAPK). This study attempted to address whether MAPK signaling and Bad phosphorylation were influenced by TGF-beta1 and, furthermore, whether these two events were involved in the antiapoptotic effect of TGF-beta1. We found a gradual activation of extracellular signal-regulated kinase 1/2 (Erk1/2) and MAPK-activated protein kinase-1 (also called Rsk1) and a concomitant increase in Bad phosphorylation at Ser(112) in mouse brains after adenovirus-mediated TGF-beta1 transduction under nonischemic and ischemic conditions induced by transient middle cerebral artery occlusion. Consistent with these effects, the ischemia-induced increase in Bad protein level and caspase-3 activation were suppressed in TGF-beta1-transduced brain. Consequently, DNA fragmentation, ischemic lesions, and neurological deficiency were significantly reduced. In cultured rat hippocampal cells, TGF-beta1 inhibited the increase in Bad expression caused by staurosporine. TGF-beta1 concentration- and time-dependently activated Erk1/2 and Rsk1 accompanied by an increase in Bad phosphorylation. These effects were blocked by U0126, a mitogen-activated protein kinase/Erk kinase 1/2 inhibitor, suggesting an association between Bad phosphorylation and MAPK activation. Notably, U0126 and a Rsk1 inhibitor (Ro318220) abolished the neuroprotective activity of TGF-beta1 in staurosporine-induced apoptosis, indicating that activation of MAPK is necessary for the antiapoptotic effect of TGF-beta1 in cultured hippocampal cells. Together, we demonstrate that TGF-beta1 suppresses Bad expression under lesion conditions, increases Bad phosphorylation, and activates the MAPK/Erk pathway, which may contribute to its neuroprotective activity.

Animals↗