[Effects and mechanism of human umbilical vein endothelial cells-derived exosomes on wound healing in diabetic rabbits].

Objective: The investigate the effects and mechanism of exosomes derived from human umbilical vein endothelial cells (HUVECs) on wound healing in diabetes rabbits. Methods: The experimental research methods were used. The primary vascular endothelial cells (VECs) and human skin fibroblasts (HSFs) were extracted from skin tissue around ulcer by surgical excision of two patients with diabetic ulcer (the male aged 49 years and the female aged 58 years) admitted to Xiangya Third Hospital of Central South University in June 2019. The cells were successfully identified through morphological observation and flow cytometry. The HUVEC exosomes were extracted by ultracentrifugation and identified successfully by morphological observation, particle size detection, and Western blotting detection. Twenty female 3-month-old New Zealand rabbits were taken to create one type 2 diabetic full-thickness skin defect wound respectively on both sides of the back. The wounds were divided into exosomes group and phosphate buffer solution (PBS) group and treated accordingly, with 20 wounds in each group, the time of complete tissue coverage of wound was recorded. On PID 14, hematoxylin-eosin staining or Masson staining was performed to observe angiogenesis or collagen fiber hyperplasia (n=20). The VECs and HSFs were co-cultured with HUVEC exosomes for 24 h to observe the uptake of HUVEC exosomes by the two kinds of cells. The VECs and HSFs were divided to exosome group treated with HUVEC exosomes and PBS group treated with PBS to detect the cell proliferation on 4 d of culture with cell count kit 8, to detect and calculate the cell migration rate at 24 and 48 h after scratch by scratch test, to detect the cell migration number at 24 h of culture with Transwell test, and to detect the mRNA expressions of nuclear factor-erythroid 2-related factor 2 (NRF2) and transcription activating factor 3 (ATF3) by real time fluorescence quantitative reverse transcription polymerase chain reaction. Besides, the number of vascular branches and vascular length were observed in the tube forming experiment after 12 h of culture of VECs (n=3). The VECs and HSFs were taken and divided into PBS group and exosome group treated as before, and NRF2 interference group, ATF3 interference group, and no-load interference group with corresponding gene interference. The proliferation and migration of the two kinds of cells, and angiogenesis of VECs were detected as before (n=3). Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, independent sample t test, and least significant difference test. Results: The time of complete tissue coverage of wound in exosome group was (17.9±1.9) d, which was significantly shorter than (25.2±2.3) d in PBS group (t=4.54, P<0.05). On PID14, the vascular density of wound in PBS group was significantly lower than that in exosome group (t=10.12, P<0.01), and the collagen fiber hyperplasia was less than that in exosome group. After 24 h of culture, HUVEC exosomes were successfully absorbed by VECs and HSFs. The proliferative activity of HSFs and VECs in exosome group was significantly higher than that in PBS group after 4 d of culture (with t values of 54.73 and 7.05, respectively, P<0.01). At 24 and 48 h after scratch, the migration rates of HSFs (with t values of 3.42 and 11.87, respectively, P<0.05 or P<0.01) and VECs (with t values of 21.42 and 5.49, respectively, P<0.05 or P<0.01) in exosome group were significantly higher than those in PBS group. After 24 h of culture, the migration numbers of VECs and HSFs in exosome group were significantly higher than those in PBS group (with t values of 12.31 and 16.78, respectively, P<0.01). After 12 h of culture, the mRNA expressions of NRF2 in HSFs and VECs in exosome group were significantly higher than those in PBS group (with t values of 7.52 and 5.78, respectively, P<0.05 or P<0.01), and the mRNA expressions of ATF3 were significantly lower than those in PBS group (with t values of 13.44 and 8.99, respectively, P<0.01). After 12 h of culture, the number of vascular branches of VECs in exosome group was significantly more than that in PBS group (t=17.60, P<0.01), and the vascular length was significantly longer than that in PBS group (t=77.30, P<0.01). After 4 d of culture, the proliferation activity of HSFs and VECs in NRF2 interference group was significantly lower than that in PBS group and exosome group (P<0.05 or P<0.01); the proliferation activity of HSFs and VECs in ATF3 interference group was significantly higher than that in PBS group (P<0.05 or P<0.01) and significantly lower than that in exosome group (P<0.05 or P<0.01). At 24 and 48 h after scratch, the migration rates of HSFs and VECs in ATF3 interference group were significantly higher than those in PBS group (P<0.05 or P<0.01) and significantly lower than those in exosome group (P<0.05 or P<0.01). At 24 and 48 h after scratch, the migration rates of HSFs and VECs in NRF2 interference group were significantly lower than those in PBS group and exosome group (P<0.05 or P<0.01). After 24 h of culture, the migration numbers of VECs and HSFs in ATF3 interference group were significantly more than those in PBS group (P<0.05) and significantly less than those in exosome group (P<0.05 or P<0.01); the migration numbers of VECs and HSFs in NRF2 interference group were significantly less than those in PBS group and exosome group (P<0.01). After 12 h of culture, the vascular length and number of branches of VECs in NRF2 interference group were significantly decreased compared with those in PBS group and exosome group (P<0.01); the vascular length and number of branches of VECs in ATF3 interference group were significantly increased compared with those in PBS group (P<0.01) and were significantly decreased compared with those in exosome group (P<0.01). Conclusions: HUVEC exosomes can promote the wound healing of diabetic rabbits by promoting the proliferation and migration of VECs and HSFs, and NRF2 and ATF3 are obviously affected by exosomes in this process, which are the possible targets of exosome action.

Identification and characterization of m1 selective muscarinic receptor antagonists1.

A series of esters of 1,4-disubstituted tetrahydropyridine carboxylic acids (I) has been synthesized and characterized as potential m1 selective muscarinic receptor antagonists. The affinity of these compounds for the five human muscarinic receptor subtypes (Hm1-Hm5) was determined by the displacement of [3H]-NMS binding using membranes from transfected Chinese hamster ovarian cells. One of the most potent and selective compounds of this series is an analogue of I [11, R1 = (CH2)5CH3], which has an IC50 value of 27.3 nM at the m1 receptor and possesses 100-fold (m2), 48-fold (m3), 74-fold (m4), and 19-fold (m5) selectivities at the other receptors. Thus, this analogue appears to be more selective on the basis of binding than the prototypical m1 antagonist, pirenzepine. Functional data, such as the inhibition of carbachol-stimulated phosphatidylinositol hydrolysis, on selected analogues confirmed the muscarinic antagonistic properties of this chemical series.

Identification and characterization of m4 selective muscarinic antagonists.

Our interest in the area of m4 muscarinic antagonists had led us to study a series of benzoxazine isoquinolines. One of the most potent and selective compounds of this series is example 1 with an IC50 value of 90.7 nM at m4 receptors, and 72-fold (m1), 38-fold (m2), 10-fold (m3), and 82-fold (m5) more selective compared to the other receptors. The synthesis and receptor binding affinity of analogs of 1 are reported.

Potent bicyclic lactam inhibitors of thrombin: Part I: P3 modifications.

Peptidomimetic inhibitors of general structure 1 have been prepared. Optimization of the binding affinities of these compounds through variation of the P3 hydrophobic residue is described. Selected substituted bicylic lactams displayed interesting pharmacological profiles both in vitro and in vivo.

Identification of a novel, sodium-dependent, reduced glutathione transporter in the rat lens epithelium.

PURPOSE: To determine whether glutathione (GSH) transporter(s) other than the previously identified rat canalicular GSH transporter (RcGshT) is present in the lens. METHODS: Poly (A) +RNA isolated from rat and guinea pig lens cortex and epithelium was injected into Xenopus laevis oocytes. The effect of sodium removal was determined by measuring cell-associated radioactivity in lenticular epithelium or cortex mRNA injected oocytes (pretreated with acivicin to inhibit gamma glutamyltranspeptidase) after 1 hour of incubation in NaCl medium or choline chloride (Na(+)-free) medium containing tracer GSH (plus unlabeled GSH). The effect of 2 mM bromosulfophthalein-GSH (BSP-GSH) on GSH uptake in the lens epithelium and cortex in NaCl medium at two GSH concentrations also was determined. The molecular form of uptake of GSH in lens epithelial mRNA-injected oocytes was examined by high-performance liquid chromatography. Western blot analysis was performed to study the presence of RcGshT in the cortex and epithelium. RESULTS: Oocytes injected with mRNA from rat and guinea pig lens epithelium and cortex compartments expressed GSH transport. High-performance liquid chromatography confirmed that epithelial uptake was as intact GSH under conditions of inhibition of GSH synthesis with dl-buthionine sulfoximine. The mean GSH uptake (nmol/oocyte per hour) in epithelial mRNA-injected oocytes was significantly reduced (P < 0.01, n = 4 oocyte preparations) under Na(+)-free conditions compared to NaCl medium at 0.05 mM and 2 mM GSH in the medium. Uptake in cortical mRNA-injected oocytes was unaffected by Na+ removal. Lens epithelial uptake exhibited a strong inhibition by BSP-GSH at 0.05 mM (55%) and 2 mM (64%), whereas cortical uptake was unaffected by BSP-GSH. Western blot analysis identified RcGshT in the cortical and epithelial regions. CONCLUSIONS: Results from the current study provide strong evidence for the presence of a hitherto unreported Na(+)-dependent, BSP-GSH inhibitable GSH transporter in the lens epithelium, which may mediate concentrative, basolateral uptake of aqueous GSH consistent with in situ eye perfusion studies. The Na(+)-independent, BSP-GSH insensitive RcGshT may function as an apical GSH effluxer in lens epithelium and in mediating concentration gradient driven inward GSH movement by uptake-efflux in the lens cortex.

Inhibition of rat sinusoidal GSH transporter by thioethers: specificity, sidedness, and kinetics.

In isolated hepatocytes, cystathionine, methionine, and thioether analogues of methionine, cysteine, and homocysteine, including S-adenosyl derivatives, inhibited reduced glutathione (GSH) efflux. The potency of inhibition by thioethers with different S-alkyl moieties was methyl < ethyl < butyryl < aminoethyl < alpha-aminopropionyl. Inhibition of GSH efflux by cystathionine from hepatocytes that were allowed to resynthesize GSH resulted in greater repletion (30-40%) of GSH levels compared with absence of cystathionine. To address unequivocally the sidedness of inhibition, i.e., cis vs. trans, we examined the effect of cystathionine on the activity of GSH transport in Xenopus oocytes expressing the cRNA of cloned rat liver sinusoidal (RsGshT) and canalicular (RcGshT) GSH transporters. Cystathionine trans inhibited efflux of GSH and cis inhibited uptake of GSH by oocytes expressing RsGshT. Conversely, when oocytes expressing RsGshT were loaded with cystathionine, no inhibition of uptake or efflux was observed. The same structural requirement of a thioether bond to exert an inhibitory effect on GSH transport was observed in oocytes expressing RsGshT. Oocytes expressing RsGshT do not transport methionine, whereas oocytes expressing total rat liver mRNA express methionine transport. Inhibition of both GSH efflux from and uptake by oocytes expressing RsGshT exhibited a competitive type of kinetics: cystathionine increased the Michaelis constant for GSH transport (4.5 +/- 0.9 vs. 10 +/- 2.5 mM and 7.5 +/- 0.6 vs. 12.9 +/- 1.5 mM for uptake and efflux, respectively) without affecting the maximal velocity for transport. Thus thioethers such as methionine and cystathionine inhibit the transport of GSH by interacting in a competitive and specific fashion with the sinusoidal GSH transporter without themselves being transported by this carrier.

Plasma membrane and mitochondrial transport of hepatic reduced glutathione.

The tripeptide glutathione (GSH) is a key nonprotein thiol that plays multiple critical functional and regulatory roles in cells. Hepatic transport of GSH is a key process in the interorgan homeostasis of GSH. Hepatocellular GSH is available to other extrahepatic organs by its release into blood and bile through the sinusoidal and canalicular GSH carriers, respectively. Their characterization at the molecular level has been recently accomplished using the functional expression cloning strategy utilizing Xenopus laevis oocytes microinjected with the corresponding cRNA from the sinusoidal (RsGshT) and canalicular (RcGshT) clones previously isolated and identified from cDNA libraries constructed from hepatic size-fraction mRNAs expressing separately the sinusoidal and canalicular GSH transporters. These clones of 2.8 and 4.0 kb encode for proteins of 39.9 and 95.8 kD for RsGshT and RcGshT, respectively, with 3 to 5 and 6 to 10 putative membrane-spanning domains. Their tissue distribution reveals that RsGshT is exclusively found in liver, contrasting with the distribution of RcGshT, which is found in nearly all tissues examined. Cellular GSH is also found in the mitochondrial matrix at a concentration similar to that in cytosol. However, mitochondria do not synthesize their own GSH, which originates from the operation of a transport carrier localized within the inner mitochondrial membrane. Its role is critical in maintaining a functionally competent organelle and in cell viability. Expression studies in Xenopus oocytes have allowed the identification of the hepatic mitochondrial GSH carrier (RmGshT), which displays distinct functional features from both RsGshT and RcGshT, such as ATP stimulation and inhibitor specificity, suggesting that RmGshT is encoded by a gene distinct from that of the plasma membrane GSH carriers.

GSH transporters: molecular characterization and role in GSH homeostasis.

Considerable progress has been made in the last few years in the molecular identification and characterization of hepatic GSH transporter-associated polypeptides. We are now poised to determine their precise mechanisms of action and regulation at the transcriptional and post-translational level. It is also anticipated that molecular characterization of the mitochondrial GSH transporter and sodium GSH co-transporters will be accomplished in the near future. With this information, a more complete understanding of GSH/cysteine homeostasis can be achieved which can be applied to furthering the prevention and treatment of the diseases of oxidative stress, such as aging, HIV, cataract, atherosclerosis, cancer and alcoholic liver disease.

Evidence for the existence of a sodium-dependent glutathione (GSH) transporter. Expression of bovine brain capillary mRNA and size fractions in Xenopus laevis oocytes and dissociation from gamma-glutamyltranspeptidase and facilitative GSH transporters.

Our laboratory previously has shown apparent carrier-mediated glutathione (GSH) uptake across the blood-brain barrier (BBB) in two animal models. In the present study, when Xenopus oocytes were injected with bovine brain capillary mRNA expression of intact GSH, uptake was observed after 3 days. When total mRNA was converted to cDNA and subfractionated with subsequent cRNA injection into oocytes, three distinct fractions (5, 7-8, and 11-12) expressed carrier-mediated intact GSH transport. Northern blot analysis established the presence of RcGshT, the previously cloned sodium-independent hepatic canalicular transporter, only in fraction 5. GSH transport activity in fraction 7 was significantly inhibited by replacement of NaCl with choline chloride and by sulfobromophthalein-GSH, neither of which affects RcGshT. The Na(+)-dependent GSH uptake kinetics exhibited high affinity (approximately 400 micron) and low affinity (approximately 10 mM) components. Fraction 11 expressed Na(+)-independent transport of intact GSH and also contained the GGT transcript. In conclusion, we have identified three distinct sized transcripts from bovine brain capillary mRNA which express GSH transport: one fraction expresses a novel Na(+)-dependent GSH uptake which can be dissociated unequivocally from both GGT and RcGshT for the first time and which may account for uptake of GSH against its electrochemical gradient at the BBB.

Molecular characterization of a reduced glutathione transporter in the lens.

PURPOSE: To characterize glutathione (GSH) transporter in the lens. METHODS: Poly (A) +RNA isolated from bovine lens was injected into Xenopus laevis oocytes. Oocytes were incubated for 1 hour in either NaCl or sucrose medium containing tracer GSH, and cell-associated radioactivity was determined. Glutathione efflux was determined in lens mRNA injected oocytes preloaded with GSH. Relationship of lens GSH transporter to the two recently cloned sodium-independent hepatic membrane GSH transporters was studied by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses. Bovine lens mRNA also was probed for gamma glutamyl transpeptidase (GGT) by RT-PCR. RESULTS: Uptake of tracer 35S-GSH could be demonstrated in X. laevis oocytes injected with poly (A) +RNA from bovine lens. Glutathione transport was carrier mediated (Km approximately 1.3 mM) and was sodium independent. High-performance liquid chromatography confirmed that the molecular form of uptake was predominantly (> 98%) as it was for GSH. Poly (A) +RNA-injected oocytes preloaded with 16.5 nmol GSH-oocyte showed GSH efflux at a rate of 2.6 nmol/oocyte per hour. When bovine lens poly (A) +RNA was hybridized with the cDNA probe for the sodium-independent rat canalicular GSH transporter (RcGshT), the transcript for RcGshT was observed. RT-PCR confirmed the presence of RcGshT and showed the absence of rat sinusoidal GSH transporter (RsGshT) and GGT mRNA in rat lens. CONCLUSIONS: The authors have demonstrated for the first time that lens contains mRNA for RcGshT and expresses a low-affinity GSH transporter in oocytes. Glutathione efflux from the apical side of the anterior epithelium and progressive uptake, and inward efflux into cortical fibers, might be explained by expression of RcGshT alone or in combination with as yet unidentified GSH transporters.

Evidence that the rat hepatic mitochondrial carrier is distinct from the sinusoidal and canalicular transporters for reduced glutathione. Expression studies in Xenopus laevis oocytes.

Mitochondrial GSH derives from a mitochondrial transport system (RmGshT), which translocates cytosol GSH into the mitochondrial matrix. Mitochondria of oocytes, isolated 3-4 days after microinjection of total liver mRNA, expressed a RmGshT compared with water-injected oocytes. The expressed RmGshT exhibited similar functional features as reported in isolated mitochondria of rat liver such as ATP stimulation, inhibition by glutamate, and insensitivity to inhibition by sulfobromophthalein-glutathione (BSP-GSH) and S-(2,4-dinitrophenyl)glutathione (DNP-GSH). The expressed RmGshT is localized in the inner mitochondrial membrane since expression is still observed in mitoplasts prepared from total liver mRNA-injected oocytes. Fractionation of poly(A)+ RNA identified a single mRNA species of approximately 3-3.5 kilobases encoding for the RmGshT, which was stimulated by ATP and inhibited by glutamate but not by BSP-GSH or DNP-GSH. Microinjection of this fraction did not lead to expression of plasma membrane GSH transport in intact oocytes, and conversely, oocytes microinjected with cRNA for rat liver sinusoidal GSH transporter (RsGshT) or rat liver canalicular GSH transporter (RcGshT) did not express mitochondrial GSH transport activity. Thus, our results show the successful expression of the rat hepatic mitochondrial GSH carrier, which is different from RsGshT and RcGshT, and provide the strategic basis for the cloning of this important carrier.

Expression cloning of the cDNA for a polypeptide associated with rat hepatic sinusoidal reduced glutathione transport: characteristics and comparison with the canalicular transporter.

Using the Xenopus oocyte expression system, we previously identified an approximately 4-kb fraction of mRNA from rat liver that expresses sulfobromophthalein reduced glutathione S-conjugate (BSP-GSH)-insensitive and an approximately 2.5-kb fraction expressing BSP-GSH-sensitive reduced glutathione (GSH) transport. From the former, a 4.05-kb cDNA was cloned and characterized as the putative rat canalicular GSH transporter. Starting with a cDNA library constructed from the approximately 2.5-kb fraction, we have now isolated a single clone that leads to expression of a BSP-GSH- and cystathionine-inhibitable GSH transporter activity with Km approximately 3 mM characteristic of the sinusoidal GSH transporter. The cDNA for the rat sinusoidal GSH transporter-associated polypeptide (RsGshT) is 2733 bases with an open reading frame of 1059 nucleotides encoding a polypeptide of 353 amino acids (39,968 Da) with two putative membrane-spanning domains. No identifiable homologies were found in searching various data bases. An approximately 40-kDa protein is generated in in vitro translation of cRNA for RsGshT. Northern blot analysis revealed a single approximately 2.8-kb transcript in rat and human liver with negligible hybridization signal in other organs. The abundance of mRNA for RsGshT did not increase with phenobarbital treatment. Cis-inhibition by BSP-GSH and trans-inhibition by cystathionine and lack of induction by phenobarbital are characteristic of sinusoidal GSH secretion and thus indicate that RsGshT either encodes the sinusoidal GSH transporter itself or a regulatory subunit of the transporter that determines its liver-specific activity.

Expression cloning of a rat hepatic reduced glutathione transporter with canalicular characteristics.

Using the Xenopus oocyte expression system, we have previously identified an approximately 4-kb fraction of mRNA from rat liver that expresses sulfobromophthalein-glutathione (BSP-GSH)-insensitive reduced glutathione (GSH) transport (Fernandez-Checa, J., J. R. Yi, C. Garcia-Ruiz, Z. Knezic, S. Tahara, and N. Kaplowitz. 1993. J. Biol. Chem. 268:2324-2328). Starting with a cDNA library constructed from this fraction, we have now isolated a single clone that expresses GSH transporter activity. The cDNA for the rat canalicular GSH transporter (RcGshT) is 4.05 kb with an open reading frame of 2,505 nucleotides encoding for a polypeptide of 835 amino acids (95,785 daltons). No identifiable homologies were found in searching various databases. An approximately 96-kD protein is generated in in vitro translation of cRNA for RcGshT. Northern blot analysis reveals a single 4-kb transcript in liver, kidney, intestine, lung, and brain. The abundance of mRNA for RcGshT in rat liver increased 3, 6, and 12 h after a single dose of phenobarbital. Insensitivity to BSP-GSH and induction by phenobarbital, unique characteristics of canalicular GSH secretion, suggest that RcGshT encodes for the canalicular GSH transporter.

Activin enhances chondrogenesis of limb bud cells: stimulation of precartilaginous mesenchymal condensations and expression of NCAM.

The roles of activin action in chicken limb development were analyzed. Activin enhanced chondrogenesis up to five-fold in a concentration-dependent manner in limb bud micromass cultures, with a half-maximal dose around 30 ng/ml (1.25 nM). The response of limb bud cells (stage 22/23) to activin appeared higher within 24 hr after culture. Activin increased the size of precartilaginous condensations. No corresponding increase in cell number was observed with total DNA or [3H]thymidine incorporation. Activin treatment resulted in increased expression of NCAM in precartilaginous condensations and tenascin in cartilage nodules, suggesting that the mechanism of activin is mediated by increased cell adhesion and recruitment of mesenchymal cells into condensations. Our results demonstrate a novel function of activin and imply that activin, together with other TGF beta superfamily members, is involved in the induction of limb chondrogenesis.

Expression of rat liver reduced glutathione transport in Xenopus laevis oocytes.

We have studied the expression of the hepatic GSH transport system in Xenopus laevis oocytes. Injection of rat liver poly(A)+ RNA resulted in the functional expression of the GSH transport system determined as GSH efflux from GSH loaded oocytes. Expression required 3-5 days to process the liver mRNA. Methionine, cystathionine, and sulfobromophthalein (BSP)-GSH inhibited the efflux of GSH from liver mRNA-injected oocytes according to their known cis or transactions on hepatocytes, namely BSP-GSH from inside and methionine and cystathionine from outside. The expressed hepatic GSH transport system also mediated the uptake of intact GSH into the oocyte, consistent with the bidirectional operation of this facilitative transporter. The uptake of GSH into mRNA-injected oocytes was inhibited by BSP-GSH in chloride-free conditions. Finally, two different mRNA size fractions encoded for hepatic GSH transport activity (uptake or efflux): a 2.0-2.5-kilobase size class, which expressed GSH transport (uptake or efflux) completely inhibited by BSP-GSH (compatible with sinusoidal GSH transport), and a 3.5-4.0-kilobase size class, which expressed GSH transport (uptake or efflux) not inhibited by BSP-GSH. These results demonstrate that hepatic GSH transport can be expressed in Xenopus oocytes and mRNA of two distinct sizes encode for GSH transporters.

Transluminal catheter angioplasty of abdominal aorta in Takayasu's arteritis.

Nine patients with Takayasu's arteritis and a long stenotic segment of the abdominal aorta were treated by percutaneous transluminal angioplasty (PTA). Intermittent claudication disappeared in six of seven cases, the femoral pulse reappeared in all five; ankle/arm indices increased in seven cases; elevated blood pressure normalized in seven of eight cases. Seven patients were followed for 3 to 28 months. They were all free of symptoms from the lower extremities. In three patients with or without renal artery stenosis and with hypertension, the blood pressure decreased after PTA of the abdominal aorta only. PTA may be a valuable treatment in Takayasu's arteritis and stenosis of the abdominal aorta.