Integrating network pharmacology and experimental models to investigate the efficacy and mechanism of Tiansha mixture on xerosis.

Epidermal Growth Factor Receptor Inhibitors (EGFRIs) is a common cancer therapy, but they occasionally cause severe side effects such as xerosis. Tiansha mixture (TM), a traditional Chinese medicines formulation, is develpoed to treat xerosis. This study aims to understand mechanisms of TM on xerosis. Bio-active compounds were selected from databases (TCMSP, TCM-ID, HERB, ETCM) and removed for poor oral bioavailability and low drug likeness. Then a network-based approach filtered out potential active compounds against xerosis. KEGG enrichment analysis identified PI3K/AKT and ERK/MAPK pathways, which were further verified by molecular docking. Afterwards, the effect of TM on activation of PI3K/AKT and ERK/MAPK pathways was validated in gefitinib-induced xerosis rats, where AKT-activator SC79 and MAPK-activator CrPic were also applied. Skin damage was assessed by dorsal score and HE and Tunel stainings. the levels of inflammation factors IL-6 and TNF-α in serum and skin tissue were measured by ELISA. Western blot was used to detect protein levels in the pathways. Network pharmacology identified 111 bio-active compounds from TM and 14 potential targets. Docking simulation showed apigenin, luteolin, and quercetin bio-active compounds in TM bound to IKBKG, INSR, and RAF-1 proteins. In xerosis model rats, TM mitigated xerosis damage, decreased inflammation factors, and phosphorylation of PI3K/AKT and ERK/MAPK proteins. SC79 or CrPic or their combination reversed TM's effect. The current study identified potential targets and PI3K/AKT and ERK/MAPK pathways involved in the effect of TM on xerosis, thus providing a foundation for TM clinical application.

Enhancement of Viable Adipose-Derived Stem Cells in Lipoaspirate by Buffering Tumescent with Sodium Bicarbonate.

BACKGROUND: Fat grafting is a growing field within plastic surgery. Adipose-derived stem cells (ASCs) and stromal vascular fracture (SVF) may have a role in fat graft survival. Our group previously demonstrated a detrimental effect on ASC survival by the lidocaine used in tumescent solution. Sodium bicarbonate (SB) buffers the acidity of lidocaine. The purpose of this study was to determine whether SB buffering is a practical method to reduce ASC and SVF apoptosis and necrosis seen with common lidocaine-containing tumescent solution. METHODS: Human patients undergoing bilateral liposuction for any indication were included in this study. An internally controlled, split-body design was utilized. Tumescent liposuction on one side of the body was conducted with tumescent containing lidocaine. On the opposite side, liposuction was conducted by adding SB to the tumescent. Tumescent solution and lipoaspirate pH were measured. Lipoaspirate from each side was processed for SVF isolation and ASC culture. The number of viable ASCs was counted and SVF apoptosis/necrosis was examined. RESULTS: The pH of the SB-buffered tumescent was significantly higher than that of the standard tumescent, an effect also seen in the lipoaspirate. Adipose-derived stem cell survival in the SB-buffered lipoaspirate was approximately 53% higher. However, there was no significant difference in SVF apoptosis and necrosis between the groups. CONCLUSIONS: The acidic standard tumescent solution commonly used in liposuction diminishes ASC viability from lipoaspirates. Sodium bicarbonate buffering tumescent solution can enhance ASC viability, but does not affect SVF apoptosis and necrosis. We recommend buffering tumescent with SB to potentially improve fat graft take. Our findings advocate for further research investigating mechanisms and optimal harvest techniques that maximize SVF/ASC survival and the clinical effect on overall fat graft viability.

Hyperbaric Oxygen Inhibits Reperfusion-Induced Neutrophil Polarization and Adhesion Via Plasmin-Mediated VEGF Release.

BACKGROUND: Ischemia-reperfusion (IR) injury is seen in many settings such as free flap salvage and limb replantation/revascularization. The consequences-partial/total flap loss, functional muscle loss, or amputation-can be devastating. Of the treatment options available for IR injury, hyperbaric oxygen (HBO) is the most beneficial. HBO inhibits neutrophil-endothelial adhesion through interference of CD18 neutrophil polarization in IR, a process mediated by nitric oxide. The purposes of this study were to examine the involvement of vascular endothelial growth factor (VEGF) in the beneficial HBO effect on CD18 polarization and neutrophil adhesion and investigate the effect of plasmin on VEGF expression in skeletal muscle following IR injury. METHODS: A rat gracilis muscle model of IR injury was used to evaluate the effect of VEGF in IR, with and without HBO, on neutrophil CD18 polarization and adhesion in vivo and ex vivo. Furthermore, we investigated the effects that plasmin has on VEGF expression in gracilis muscle and pulmonary tissue by blocking its activation with alpha-2-antiplasmin. RESULTS: HBO treatment following IR injury significantly decreased neutrophil polarization and adhesion ex vivo compared with the IR group. Anti-VEGF reversed the beneficial HBO effect after IR with polarization and adhesion. In vivo adhesion was also increased by anti-VEGF. HBO treatment of IR significantly increased the VEGF protein in both gracilis and pulmonary vasculature. Alpha-2-antiplasmin significantly reversed the HBO-induced increase of VEGF in gracilis muscle. CONCLUSIONS: These results suggest that HBO inhibits CD18 polarization and neutrophil adhesion in IR injury through a VEGF-mediated pathway involving the extracellular matrix plasminogen system.

Tumescent Liposuction without Lidocaine.

BACKGROUND: Our previous study demonstrated that lidocaine has a negative impact on adipose-derived stem cell (ASC) survival. Currently for large-volume liposuction, patients often undergo general anesthesia; therefore, lidocaine subcutaneous anesthesia is nonessential. We hypothesized that removing lidocaine from tumescent might improve stromal vascular fraction (SVF) and ASC survival from the standard tumescent with lidocaine. Ropivacaine is also a commonly used local anesthetic. The effect of ropivacaine on ASC survival was examined. METHODS: Adults who underwent liposuction on bilateral body areas were included (n = 10). Under general anesthesia, liposuction on 1 area was conducted under standard tumescent with lidocaine. On the contralateral side, liposuction was conducted under the modified tumescent without lidocaine. Five milliliters of lipoaspirate were processed for the isolation of SVF. The adherent ASCs were counted after 24 hours of SVF culture. Apoptosis and necrosis of SVF cells were examined by Annexin/propidium iodide staining and analyzed by flow cytometry. RESULTS: Average percentage of live SVF cells was 68.0% ± 4.0% (28.5% ± 3.8% of apoptosis and 3.4% ± 1.0% of necrosis) in lidocaine group compared with 86.7% ± 3.7% (11.5% ± 3.1% of apoptosis and 1.8% ± 0.7% of necrosis) in no-lidocaine group (P = 0.002). Average number of viable ASC was also significantly lower (367,000 ± 107) in lidocaine group compared with that (500,000 ± 152) in no-lidocaine group (P = 0.04). No significant difference was found between lidocaine and ropivacaine on ASC cytotoxicity. CONCLUSIONS: Removing lidocaine from tumescent significantly reduced SVF and ASC apoptosis in the lipoaspirate. We recommend tumescent liposuction without lidocaine, particularly if patient's lipoaspirate will be used for fat grafting.

Elimination of reperfusion-induced microcirculatory alterations in vivo by adipose-derived stem cell supernatant without adipose-derived stem cells.

BACKGROUND: In the present study, the authors hypothesized that adipose-derived stem cells in cell culture may secrete multiple cytokines in the supernatant, which might have a significant impact in vivo on the reperfusion-induced microcirculatory alterations and endothelial dysfunction. METHODS: Fat tissue was surgically harvested from rat flanks and processed for adipose-derived stem cell isolation; cells (1 × 10(6)) were subcultured for 3, 6, 9, and 12 days without passage. The postcultivated medium was harvested with medium change every 3 days. After centrifugation, the supernatant was collected and stored at -20°C. Supernatant collected on day 9 was analyzed for eight oxidative stress cytokines by an enzyme-linked immunosorbent assay strip. The effect of the supernatant on the reperfusion-induced microcirculatory alterations was examined in the vascular pedicle of isolated rat cremaster muscles subjected to 4 hours of ischemia followed by 2 hours of reperfusion. RESULTS: Enzyme-linked immunosorbent assay results demonstrated that adipose-derived stem cells produced several highly expressed cytokines in the supernatant. The average concentration of interleukin-6, in particular, was 5-fold higher compared with control. The reperfusion-induced vasospasm, arteriole stagnation, and the capillary no-reflow that often appear in the early phase of reperfusion were eliminated by adipose-derived stem cell supernatant. CONCLUSIONS: Adipose-derived stem cells in cell culture display cytokine secretory properties that enable the cells to act through paracrine signaling. The supernatant even without cells could be used as a paracrine agent to interfere with the reperfusion-induced microcirculatory alterations and endothelial dysfunction.

Lidocaine-induced ASC apoptosis (tumescent vs. local anesthesia).

BACKGROUND: The purpose for the present study was to determine which anesthetic method, local anesthesia versus tumescent, is superior for liposuction in terms of adipose-derived stem cell (ASC) survival in lipoaspirate; which component, lidocaine versus lidocaine with epinephrine, in anesthetic solutions could affect ASC survival; and which mechanism, necrosis versus apoptosis, is involved in lidocaine-induced ASC death. METHODS: Human lipoaspirates were harvested using standard liposuction technique. Individuals scheduled for liposuction on bilateral body areas gave consent and were included in the study. On one area, liposuction was conducted under local anesthesia with lidocaine/epinephrine. On the contralateral area, liposuction was accomplished with tumescent wetting solution containing lidocaine/epinephrine. Lipoaspirates were processed for the isolation of stromal vascular fraction (SVF). ASC survival was determined by the number of adherent ASCs after 24 h of SVF culture. Lidocaine dose-response (with or without epinephrine) on cultured ASCs was examined. Lidocaine-induced ASC apoptosis and necrosis was determined by Annexin V-FITC/Propidium Iodide (PI) assay and analyzed by flow cytometry. RESULTS: All of the participants were female adults. The average age was 45 ± 4.0 years (±SEM) and the average BMI was 28 ± 1.0 (±SEM). Lipoaspirate samples (n = 14) treated by local anesthesia (n = 7/group) or tumescent anesthesia (n = 7/group) were investigated. Liposuction sites were located in the hip or thigh. The average number of adherent ASCs was 1,057 ± 146 k in the local anesthesia group, which was significantly lower than the 1,571 ± 111 k found in the tumescent group (P = 0.01). ASC survival was significantly lower in the lidocaine group and in a dose-dependent manner as compared to the correspondent PBS controls (P < 0.05 or P < 0.01). ASC survival was significantly lower in both the lidocaine and lidocaine with epinephrine groups when compared to PBS controls. Annexin/PI assay showed that ASC apoptosis (but not necrosis) in the lidocaine group was significantly higher than that in the corresponding PBS control (P = 0.026). CONCLUSIONS: Tumescent anesthesia is the superior method for liposuction with respect to ASC preservation compared to local anesthesia. Lidocaine could cause significant ASC apoptosis.

The effect of lipoaspirates cryopreservation on adipose-derived stem cells.

BACKGROUND: Autologous fat grafting has gained popularity, particularly with the discovery of adipose-derived stem cells (ADSC). The possibility of freezing lipoaspirates (LA) for later use has intriguing clinical potential. However, the effect of LA cryopreservation on ADSC is unclear. OBJECTIVES: The authors explore the effect of LA cryopreservation on ADSC viability. METHODS: Human LA (n = 8) were harvested using a standard technique. Lipoaspirate samples were either processed immediately as fresh LA (A) or stored at -20°C and then at -80°C for 30 days with (B) or without (C) freezing medium. Stromal vascular fraction (SVF) was separated from adipocytes and either cultured to obtain purified ADSC or processed for the isolation of 3 distinct ADSC subpopulations (CD90(+)/CD45(-), CD105(+)/CD45(-), and CD34(+)/CD31(-)). Apoptosis and necrosis were determined by an annexin V/propidium iodide assay and quantified by flow cytometry. The capability of ADSC for long-term proliferation and differentiation was also examined. RESULTS: There were no significant differences in the apoptosis and necrosis of adipocytes, SVF, or ADSC between groups A and B. However, cell viability in SVF and ADSC was significantly compromised in group C as compared with group B (P < .01) due to higher ADSC apoptosis but not necrosis. The viable ADSC isolated from fresh or frozen LA were cultured for more than 20 passages and demonstrated similar patterns and speed of proliferation with strong capability to differentiate, evidenced by cell doubling time and positive staining with Oil Red O (Sigma-Aldrich, St Louis, Missouri) and alkaline phosphatase. CONCLUSIONS: Lipoaspirates cryopreservation had a significant impact on ADSC apoptosis but not on ADSC necrosis, proliferation, or differentiations. Freezing medium provides significant protection against ADSC apoptosis.

Analysis for apoptosis and necrosis on adipocytes, stromal vascular fraction, and adipose-derived stem cells in human lipoaspirates after liposuction.

BACKGROUND: Adipose-derived stem cells have become the most studied adult stem cells. The authors examined the apoptosis and necrosis rates for adipocyte, stromal vascular fraction, and adipose-derived stem cells in fresh human lipoaspirates. METHODS: Human lipoaspirate (n = 8) was harvested using a standard liposuction technique. Stromal vascular fraction cells were separated from adipocytes and cultured to obtain purified adipose-derived stem cells. A panel of stem cell markers was used to identify the surface phenotypes of cultured adipose-derived stem cells. Three distinct stem cell subpopulations (CD90/CD45, CD105/CD45, and CD34/CD31) were selected from the stromal vascular fraction. Apoptosis and necrosis were determined by annexin V/propidium iodide assay and analyzed by flow cytometry. RESULTS: The cultured adipose-derived stem cells demonstrated long-term proliferation and differentiation evidenced by cell doubling time and positive staining with oil red O and alkaline phosphatase. Isolated from lipoaspirates, adipocytes exhibited 19.7 ± 3.7 percent apoptosis and 1.1 ± 0.3 percent necrosis; stromal vascular fraction cells revealed 22.0 ± 6.3 percent of apoptosis and 11.2 ± 1.9 percent of necrosis; stromal vascular fraction cells had a higher rate of necrosis than adipocytes (p < 0.05). Among the stromal vascular fraction cells, 51.1 ± 3.7 percent expressed CD90/CD45, 7.5 ± 1.0 percent expressed CD105/CD45, and 26.4 ± 3.8 percent expressed CD34/CD31. CD34/CD31 adipose-derived stem cells had lower rates of apoptosis and necrosis compared with CD105/CD45 adipose-derived stem cells (p < 0.05). CONCLUSIONS: Adipose-derived stem cells had a higher rate of apoptosis and necrosis than adipocytes. However, the extent of apoptosis and necrosis was significantly different among adipose-derived stem cell subpopulations.

Novel biosensor-based microarray assay for detecting rs8099917 and rs12979860 genotypes.

AIM: To evaluate a novel biosensor-based microarray (BBM) assay for detecting rs12979860 and rs8099917 genotypes. METHODS: Four probes specific for rs8099917C/T or rs12979860G/T detection and three sets of quality control probes were designed, constructed and arrayed on an optical biosensor to develop a microarray assay. Two sets of primers were used in a one tube polymerase chain reaction (PCR) system to amplify two target fragments simultaneously. The biosensor microarray contained probes that had been sequenced to confirm that they included the rs8099917C/T or rs12979860G/T alleles of interest and could serve as the specific assay standards. In addition to rehybridization of four probes of known sequence, a total of 40 clinical samples collected from hepatitis C seropositive patients were also tested. The target fragments of all 40 samples were amplified in a 50 µL PCR system. Ten µL of each amplicon was tested by BBM assay, and another 40 µL was used for sequencing. The agreement of the results obtained by the two methods was tested statistically using the kappa coefficient. The sensitivity of the BBM assay was evaluated using serial dilutions of ten clinical blood samples containing 10(3)-10(4) white cells/µL. RESULTS: As shown by polyacrylamide gel electrophoresis, two target segments of the interleukin 28B-associated polymorphisms (SNPs) were successfully amplified in the one-tube PCR system. The lengths of the two amplified fragments were consistent with the known length of the target sequences, 137 and 159 bps. After hybridization of the PCR amplicons with the probes located on the BBM array, the signals of each allele of both the rs8099917 SNPs and rs12979860 SNPs were observed simultaneously and were clearly visible by the unaided eye. The signals were distinct from each other, could be interpreted visually, and accurately recorded using an ordinary digital camera. To evaluate the specificity of the assay, both the plasmids and clinical samples were applied to the microarray. First, 30 PCR amplicons of the various SNP alleles were hybridized on the BBM microarray. Full agreement between plasmids and the BBM assay was observed, with 30/30 correct matches (100%). The kappa value for the BBM assay with plasmids was 1.00 (P < 0.05). For the 40 clinical blood samples, the BBM assay hybridization and direct sequencing results were compared for each amplicon. For patient blood samples, agreement was 28/28 for rs8099917T/T, 9/11 for rs8099917T/G, 1/1 for rs8099917G/G, 24/24 for rs12979860C/C, 11/14 for rs12979860C/T, and 2/2 for rs12979860T/T. Only five clinical samples of amplicon assay and direct sequencing results were discordant and heterozygotes: 2/11 rs8099917T/G and 3/14 rs12979860C/T. The agreement of outcomes between BBM assay and direct sequencing for the detection of rs8099917 and rs12979860 was 95% and 92.5%, respectively; and the corresponding kappa values were 0.88 and 0.85 (A kappa value > 0.75 was defined as substantial agreement). The BBM assay and sequencing had similar specificities for detection and identification of the two SNPs and their alleles. The sensitivity evaluation showed that the BBM assay could detect and identify SNP sequences present in blood samples containing as few as 10(2) white blood cells/µL. CONCLUSION: This biosensor microarray assay was highly specific, sensitive, rapid and easy to perform. It is compatible with clinical practice for detection of rs8099917 and rs12979860.

Nitrite attenuates ischemia-reperfusion-induced microcirculatory alterations and mitochondrial dysfunction in the microvasculature of skeletal muscle.

BACKGROUND: Recently, nitrite has been rediscovered as a physiologically relevant storage reservoir of nitric oxide in blood and it can readily be converted to nitric oxide under hypoxic and acidic conditions. In this study, the authors evaluated the therapeutic efficacy of nitrite on reperfusion-induced microcirculatory alterations and mitochondrial dysfunction in the microvasculature of skeletal muscle. METHODS: The authors used a vascular pedicle isolated rat cremaster model that underwent 4 hours of warm ischemia followed by 2 hours or 17 hours of reperfusion. At 5 minutes before reperfusion, normal saline, sodium nitrite (0.20 µM/minute/kg), or nitrite mixed with 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (potassium salt) (0.2 mg/minute/kg) was infused into the microcirculation of ischemic cremaster by means of intraarterial infusion. Ischemia-reperfusion-induced microcirculatory alterations were measured after 2 hours of reperfusion. Microvasculature of the cremaster muscle including the vascular pedicle was harvested to determine the mitochondrial dysfunction. The blood concentration of methemoglobin was also measured to determine the toxicity of nitrite. RESULTS: The authors found that nitrite significantly attenuated ischemia-reperfusion-induced vasoconstriction, arteriole stagnation, and capillary no-reflow in the early phase of reperfusion and the depolarization of mitochondrial membrane potential and cytochrome c release in the late phase of reperfusion. Nitrite-induced protection was significantly blocked by a nitric oxide scavenger (potassium salt). The methemoglobin results showed that the doses of nitrite we used in the present study were safe. CONCLUSION: The supplementation of a low dose of nitrite, directly into the microcirculation of ischemic muscle through local intraarterial infusion, significantly attenuated ischemia-reperfusion-induced microcirculatory alterations in vivo and mitochondrial dysfunction in vitro in the microvasculature of skeletal muscle.

Melatonin attenuates I/R-induced mitochondrial dysfunction in skeletal muscle.

BACKGROUND: Our recent studies have shown that ischemia/reperfusion (I/R) produces significant necrosis and apoptosis in the cells of skeletal muscle. Our previous studies also demonstrated that melatonin provides significant protection against superoxide generation, endothelial dysfunction, and cell death in the skeletal muscle after I/R. Mitochondria are essential for cell survival, because of their roles as ATP producers as well as regulators of cell death. However, the efficacy of melatonin on I/R-induced mitochondrial dysfunction in the skeletal muscle in vivo has not been demonstrated in the literature. MATERIALS AND METHODS: Vascular pedicle isolated rat gracilis muscle model was used. After 4 h of ischemia followed by 24 h of reperfusion, gracilis muscle was harvested, and mitochondrial as well as cytosolic fractions were isolated. Mitochondrial dysfunction was determined by the alteration of mitochondrial membrane potential and the release of the proapoptotic protein, cytochrome c. Three groups were designed; sham I/R, I/R-V (I/R with vehicle), and I/R-Mel (I/R with melatonin). Melatonin or vehicle was given intravenously 10 min prior to reperfusion and 10 min after reperfusion. RESULTS: We found that the capability of uptake of fluorescent JC-1 dye in skeletal muscle cells was substantially improved in I/R-Mel group compared with I/R-V group. Melatonin significantly inhibited the outflow of cytochrome c from mitochondria to cytoplasm, which was demonstrated in the I/R-V group. CONCLUSIONS: Melatonin significantly attenuates I/R-induced mitochondrial dysfunction, such as the depolarization of mitochondrial membrane potential and the release of the proapoptotic protein, cytochrome c, from the mitochondria.

Ischemia-reperfusion-induced apoptotic endothelial cells isolated from rat skeletal muscle.

BACKGROUND: The purpose of the present study was to investigate ischemia-reperfusion-induced apoptosis and necrosis in endothelial cells isolated from skeletal muscle. METHODS: A vascular pedicle isolated rat gracilis muscle model was used. After surgical preparation, clamps were applied to the vascular pedicle to create 4 hours of ischemia and released for reperfusion (ischemia-reperfusion group, n = 9). Clamping was omitted in sham ischemia-reperfusion rats (sham ischemia-reperfusion group, n = 9). The muscle samples were harvested after 20 hours of reperfusion for the process of cell isolation. One hundred thousand cells from each sample were stained by monoclonal anti-CD146-fluorescein (a principal marker for mature endothelial cells), Annexin V-PE, or 7-aminoactinomycin D to detect and quantify apoptotic and necrotic cells. Twenty thousand cells from each sample were scanned and analyzed by flow cytometry. RESULTS: The average +/- SEM of CD146-fluorescein-positive cells was 20.0 +/- 2.9 percent, suggesting that these cells might be endothelial cells from the muscle microvasculature. In the population of gated CD146-fluorescein-positive cells, the average percentage of apoptotic cells (stained by Annexin V-PE) was 15.9 +/- 2.2 percent in the sham ischemia-reperfusion group and 33.5 +/- 5.3 percent in the ischemia-reperfusion group (p < 0.01), the average percentage of necrotic/apoptotic cells (stained by both 7-aminoactinomycin D and Annexin V-PE) was 17.8 +/- 4.1 percent in the sham ischemia-reperfusion group and 39.2 +/- 3.1 percent in the ischemia-reperfusion group (p < 0.01). CONCLUSIONS: Given the results of the present study, the authors hypothesize that the endothelial cells lining microscopic blood vessels are among the major contributors to ischemia-reperfusion-induced cell apoptosis and necrosis detected from rat skeletal muscle.

Ischemia/reperfusion-induced necrosis and apoptosis in the cells isolated from rat skeletal muscle.

Necrosis was considered to be the solo mechanism for ischemia/reperfusion (I/R)-induced cell death. Recent evidence from I/R models of the heart, liver, kidney, and brain indicates that apoptosis is a major contributor to I/R-induced cell death. However, evidence of I/R-induced apoptosis in skeletal muscle is sparse and divided. The purpose for the present study was to investigate I/R-induced necrosis and apoptosis in the cells isolated from rat skeletal muscle. A rat gracilis muscle model was used. After surgical preparation, clamps were applied on the vascular pedicle to create 4 h of ischemia and released for 24 h of reperfusion (I/R, n = 10). Clamping was omitted in sham I/R rats (sham I/R, n = 10). The muscle samples were harvested after 24 h of reperfusion for the process of cell isolation. Cells were stained by Propidium Iodide (PI) or Annexin V-FITC or both. Twenty thousand cells from each muscle sample were scanned and analyzed by flow cytometry. The average percentage of live cells was 45 +/- 2% in the I/R group versus 65 +/- 3% in the sham I/R group (p < 0.01). The average percentage of necrotic cells was 18 +/- 1% in I/R versus 12 +/- 1% in sham I/R (p < 0.01). The average percentage of apoptotic cells was 40 +/- 3% in I/R versus 27 +/- 3% in sham I/R (p < 0.01). Our results clearly demonstrated that I/R not only causes necrosis, but also accelerates apoptosis in the cells isolated from rat skeletal muscle.

Effects of supplementation of BH4 after prolonged ischemia in skeletal muscle.

PURPOSE: To determine whether the supplementation of tetrahydrobiopterin (BH(4), an essential cofactor of nitric oxide synthase; NOS) could attenuate endothelial dysfunction and improve NOS activity and cell viability in skeletal muscle after ischemia/reperfusion (I/R). METHODS: A vascular pedicle isolated rat cremaster muscle model was used. Cremaster muscles were subjected to 4 h of ischemia followed by 2 h of reperfusion. Rats were given either normal saline or BH(4) by intravenous injection at 1 min prior to reperfusion. After reperfusion, average arteriole diameter, capillary perfusion, endothelial-dependent/-independent vasodilatation, NOS activity, and muscle cell viability were evaluated. RESULTS: Supplementation of BH(4) prior to reperfusion significantly attenuated reperfusion-induced vasoconstriction, poor capillary perfusion, and endothelial dysfunction and enhanced cNOS activity and slightly improved cell viability in the skeletal muscle after I/R. CONCLUSION: Supplementation of BH(4) during reperfusion provided a significant protection against I/R injury in rat skeletal muscle.

Melatonin reduces ischemia/reperfusion-induced superoxide generation in arterial wall and cell death in skeletal muscle.

The purpose of this study was to determine the effect of melatonin on superoxide generation in arterial wall at an early phase of reperfusion and on endothelial dysfunction of microvasculature and cell viability of cremaster muscle at late phase of reperfusion (24 hr) after prolonged ischemia. Bilateral vascular pedicles which supply blood flow to the cremaster muscle were exposed. After surgical preparation, microvascular clamps were applied on the right iliac, femoral and spermatic arteries to create 4 hr of ischemia in both feeding vessels and the unexposed cremaster muscle. The vascular clamping was omitted on the left iliac, femoral and spermatic arteries and served as an internal control. Melatonin or Vehicle was via by intravenous injection at 10 min prior to reperfusion and 10 min after reperfusion. In the first experiment, the vascular pedicle was harvested after reperfusion to measure superoxide generation in real time by lucigenin-derived chemiluminescence. In the second experiment, endothelial-dependent and -independent vasodilatation was examined in the terminal arteriole of cremaster muscle which was then harvested to examine cell viability by WST-1 assay on day 2. Superoxide generation in arterial wall peaked at first 5-min of reperfusion and declined to near baseline after 60 min of reperfusion. Melatonin treatment significantly reduced superoxide generation in arterial walls and improved cell viability in cremaster muscles. Melatonin treatment also significantly reduced microvascular endothelial dysfunction which was still observable in the microcirculation of cremaster muscle after 24 hr of reperfusion. Melatonin reduces superoxide generation in the early phase of reperfusion resulting in attenuating endothelial dysfunction and muscle cell death in the late phase of reperfusion.

Host cell responses induced by hepatitis C virus binding.

Initiation of hepatitis C virus (HCV) infection is mediated by docking of the viral envelope to the hepatocyte cell surface membrane followed by entry of the virus into the host cell. Aiming to elucidate the impact of this interaction on host cell biology, we performed a genomic analysis of the host cell response following binding of HCV to cell surface proteins. As ligands for HCV-host cell surface interaction, we used recombinant envelope glycoproteins and HCV-like particles (HCV-LPs) recently shown to bind or enter hepatocytes and human hepatoma cells. Gene expression profiling of HepG2 hepatoma cells following binding of E1/E2, HCV-LPs, and liver tissue samples from HCV-infected individuals was performed using a 7.5-kd human cDNA microarray. Cellular binding of HCV-LPs to hepatoma cells resulted in differential expression of 565 out of 7,419 host cell genes. Examination of transcriptional changes revealed a broad and complex transcriptional program induced by ligand binding to target cells. Expression of several genes important for innate immune responses and lipid metabolism was significantly modulated by ligand-cell surface interaction. To assess the functional relevance and biological significance of these findings for viral infection in vivo, transcriptional changes were compared with gene expression profiles in liver tissue samples from HCV-infected patients or controls. Side-by-side analysis revealed that the expression of 27 genes was similarly altered following HCV-LP binding in hepatoma cells and viral infection in vivo. In conclusion, HCV binding results in a cascade of intracellular signals modulating target gene expression and contributing to host cell responses in vivo. Reprogramming of cellular gene expression induced by HCV-cell surface interaction may be part of the viral strategy to condition viral entry and replication and escape from innate host cell responses.

Microcirculatory effects of melatonin in rat skeletal muscle after prolonged ischemia.

The purpose of this study was to determine microcirculatory effects and response of nitric oxide synthase (NOS) to melatonin in skeletal muscle after prolonged ischemia. A vascular pedicle isolated rat cremaster muscle model was used. Each muscle underwent 4 hr of zero-flow warm ischemia followed by 2 hr of reperfusion. Melatonin (10 mg/kg) or saline as a vehicle was given by intraperitoneal injection at 30 min prior to reperfusion and the same dose was given immediately after reperfusion. After reperfusion, microcirculation measurements including arteriole diameter, capillary perfusion and endothelial-dependent and -independent vasodilatation were performed. The cremaster muscle was then harvested to measure endothelial NOS (eNOS) and inducible NOS (iNOS) gene expression and enzyme activity. Three groups of rats were used: sham-ischemia/reperfusion (I/R), vehicle + I/R and melatonin + I/R. As compared with vehicle + I/R group, administration of melatonin significantly enhanced arteriole diameter, improved capillary perfusion, and attenuated endothelial dysfunction in the microcirculation of skeletal muscle after 4 hr warm ischemia. Prolonged warm ischemia followed by reperfusion significantly depressed eNOS gene expression and constitutive NOS activity and enhanced iNOS gene expression. Administration of melatonin did not significantly alter NOS gene expression or activity in skeletal muscle after prolonged ischemia and reperfusion. Melatonin provided a significant microvascular protection from reperfusion injury in skeletal muscle. This protection is probably attributable to the free radical scavenging effect of melatonin, but not to its anti-inflammatory effect.

Acute microvascular action of vascular endothelial growth factor in skeletal muscle ischemia/reperfusion injury.

BACKGROUND: The purpose of this study was to investigate the acute action of vascular endothelial growth factor (VEGF) in the microcirculation of skeletal muscle subject to ischemia/reperfusion in vivo and to determine the role of nitric oxide synthase in VEGF-induced microvascular protection. METHODS: A vascular pedicle isolated rat cremaster muscle model coupled with local intraarterial infusion technique was used. Each muscle underwent 4 hours of zero-flow warm ischemia followed by 2 hours of reperfusion. Femoral artery cannulation was performed before reperfusion. The infusate was administered by continuous infusion into the arterial tree of the muscle beginning at 1 minute before reperfusion and at the rate of 0.1 ml/hour throughout the entire reperfusion period. Three groups were designed: (1) the ischemia/reperfusion group, with infusion normal saline; (2) the VEGF plus ische-mia/reperfusion group, with infusion of recombinant human VEGF165 protein; and (3) the L-NA plus VEGF plus ischemia/reperfusion group, with infusion of N-nitro-L-arginine (L-NA; a nonselective nitric oxide synthase antagonist) mixed with VEGF165 protein. After 2 hours of reperfusion, microcirculation measurements including arteriole diameter, capillary perfusion, and endothelium-dependent and endothelium-independent vasodilatation were performed. The muscle was harvested and processed for reverse-transcriptase polymerase chain reaction for measuring eNOS and endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) gene expression. RESULTS: Reperfusion caused significant microvascular alterations including vasoconstriction, poor capillary perfusion, and endothelial dysfunction in the skeletal muscle. These alterations were significantly attenuated by intraarterial infusion of VEGF during reperfusion, but the beneficial effect of VEGF was reduced significantly by coadministration of L-NA. Reverse-transcriptase polymerase chain reaction study revealed that ischemia/reperfusion depressed eNOS mRNA expression but enhanced iNOS mRNA expression. Intraarterial infusion of VEGF during reperfusion amplified mRNA expression of eNOS but not of iNOS. CONCLUSIONS: Local intraarterial infusion of VEGF produced significant microvascular protection from skeletal muscle ischemia/reperfusion injury. The VEGF-induced enhancement of eNOS may play an important mechanistic role.

NOS upregulation attenuates vascular endothelial dysfunction in the late phase of ischemic preconditioning in skeletal muscle.

Previously, we have demonstrated a late phase protection of ischemic preconditioning in the microcirculation of cremaster muscle. This microvascular protection was blocked by a non-specific NOS inhibitor. The purpose of present study was to evaluate endothelial function in the terminal arteriole of cremaster muscle after 24-h of ischemic preconditioning followed by 4-h warm ischemia and to evaluate eNOS and iNOS gene and protein expression at 24 h after ischemic preconditioning in the cremaster muscle. A vascular pedicle isolated cremaster muscle in male SD rats underwent 45-min of ischemic preconditioning and 24 h later, 4-h of warm ischemia followed by reperfusion. Endothelial-dependent and -independent vasodilatation was evaluated on day 2 after 4-h ischemia and 60-min of reperfusion. Cremaster muscles were harvested at 24 h after ischemic preconditioning for measuring of eNOS and iNOS gene expression by reverse transcriptase polymerase chain reaction (RT-PCR) and protein expression by western blotting analysis. We found that IPC significantly attenuated endothelial dysfunction induced by 4-h warm ischemia and reperfusion. The expression of eNOS and iNOS mRNA shown a 229% and 135% increase respectively in IPC treated cremaster muscles as compared to normal cremaster muscles (P<0.05). The expression of eNOS and iNOS protein exhibited a 133% and 148% increase respectively in IPC treated cremaster muscles as compared to normal cremaster muscles (P<0.05). There was no statistically significant difference between normal cremaster muscle and sham IPC treated cremaster muscle. The results suggest that IPC preventing vascular endothelial dysfunction from ischemia/reperfusion injury may be due to the enhanced NOS expression. These results combined with the results from our previous studies suggest that IPC-induced microvascular protection in the skeletal muscle may act through a NOS-dependent mechanism.