Xin-Hua Fang

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

Abstract:  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.

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.

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

Abstract:  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.

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.

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 &mgr;M/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.

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-aminoactinomycinD 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.

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.

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 × 106) 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.

The impact of short-term refrigeration of human lipoaspirate on adipose-derived stem cells and adipocytes

Both liposuction and fat grafting are popular procedures in aesthetic surgery. Current clinical practice is that fat grafting has to be conducted immediately after liposuction in the concern that cell death in lipoaspirate could increase as the storage time is prolonged. However, there is no scientific data in the literature either to support or oppose that concern. It has been a strong desire of both surgeons and patients to be able to preserve lipoaspirate for potential future applications. In our previous studies, the viability of adipose-derived stem cells (ASCs) and adipocytes in both fresh and deep-frozen lipoaspirates has been examined and cell apoptosis and necrosis have been quantified by Annexin-V/PI assay and analyzed by flow cytometer. The purpose for the present study was to determine the impact of short-term refrigeration of lipoaspirate on ASCs and adipocytes. Two quantitated functional assays were selected to determine cell viability based on the concept that viable cell does not necessarily mean functional, but the functional cell must be viable. Human lipoaspirates were harvested using standard technique. Each lipoaspiratewas divided into 5 portions with 5 ml of each. The fresh portion was processed immediately and served as control. Other portions were stored in the refrigerator (2e8 C) and processed at 24 h, 48 h, 72 h or 96 h later respectively. The viability of ASCs was determined by the number of ASC after 24 h culture of stromal vascular fraction (SVF). The viability of adipocytes was assessed by glycerol-3-phosphate dehydrogenase (G3PDH) activitywhich is widely used to evaluate the biosynthesis of fat in adipocytes. The sterility of lipoaspirate was assessed by bacterial culture using LB Agar powder and inoculating loop.

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.