富血小板血浆凝胶制备及对大鼠皮瓣成活的作用分析

目的寻找制备富血小板血浆(platelet-rich plasma,PRP)的最佳离心方案,并制备PRP凝胶用于皮下注射促进大鼠皮瓣成活,探讨其有效性及作用机制。方法取12周龄健康Wistar大鼠72只,体重250～300 g。于24只大鼠心脏取动脉血8～10 mL/只,分别采用3种离心方法制备PRP。A组:以200×g离心15 min、500×g离心10 min;B组:以312×g离心10 min、1 248×g离心10 min;C组:以200×g离心15 min,200×g离心10 min。在PRP制作过程中取各组全血、PRP及贫血小板血浆(platelet-poor plasma,PPP)行血小板计数,根据血小板计数结果选择最佳离心方案,并取对应的PRP、PPP及第1次离心后血清,采用ELISA法测定PDGF-BB和TGF-β1浓度;并制备PRP及PPP凝胶。于48只大鼠背部制备大小为11 cm×3 cm的皮瓣,随机分为3组(n=16):PRP组每只大鼠皮瓣下注射100 mL PRP凝胶,PPP组同法注射100 mL PPP凝胶,对照组不作处理。术后大体观察皮瓣成活情况,7 d后取材计算成活率;组织学观察计数炎性细胞,免疫组织化学染色计数微血管;于术后8、24 h,3、7 d行实时荧光定量PCR检测VEGF、EGF、PDGF-AA和PDGF-BB mRNA表达。结果血小板计数结果示,A组血小板浓缩倍数最高,为最佳制备方法。A组PRP中TGF-β1、PDGF-BB浓度明显高于血清及PPP(P<0.05)。与对照组相比,术后PRP组伤口无脓性分泌物;PRP组皮瓣成活率为61.2%±9.1%,与PPP组35.8%±11.3%及对照组28.0%±5.4%比较,差异有统计学意义(P<0.05)。PRP组炎性细胞计数较PPP组及对照组明显减少,微血管计数明显高于其余两组,差异均有统计学意义(P<0.05)。实时荧光定量PCR检测示与PPP组及对照组比较,PRP组VEGF及PDGF-BB mRNA术后均高表达,而EGF mRNA仅在术后24 h内呈高表达,PDGF-AA mRNA在3 d后开始高表达。术后3 d及7 d PRP组PDGF-AA、8 h PDGF-BB、24 h及3 d VEGF、24 h EGF的mRNA相对表达量与其他两组比较,差异有统计学意义(P<0.05)。结论以200×g离心15 min、500×g离心10 min是制备PRP的最佳离心方案。PRP凝胶可通过调节血管生发相关基因促进大鼠皮瓣成活。     

富血小板血浆制备套装的评估研究

目的通过分析富血小板血浆(platelet-rich plasma,PRP)中血小板、白细胞和生长因子的浓度,计算回收率和富集系数,并做相关性分析,探讨PRP制备套装的实用性和稳定性。方法取30例符合纳入标准的自愿者自愿捐赠的外周血各40mL,应用山东威高集团医用高分子制品股份有限公司的PRP制备套装制备PRP各4mL。全自动血液分析仪计数全血和PRP中血小板和白细胞浓度,并计算血小板或白细胞回收率及富集系数;并分别测定男、女自愿者血小板及白细胞浓度。ELISA法定量分析激活后全血及PRP中PDGF、TGF-β、VEGF的浓度。结果全血和PRP中血小板浓度分别为(131.40±29.44)×109/L和(819.47±136.32)×109/L,比较差异有统计学意义(t=—27.020,P=0.000);PRP中血小板回收率为60.85%±8.97%,富集系数为6.40±1.06。全血和PRP中白细胞浓度分别为(5.57±1.91)×1012/L和(32.20±10.42)×1012/L,比较差异有统计学意义(t=—13.780,P=0.000);PRP中白细胞回收率为58.30%±19.24%,富集系数为6.10±1.93。PRP中血小板浓度和白细胞浓度分别与全血中血小板浓度(r=0.652,P=0.000)和白细胞浓度(r=0.460,P=0.011)成正相关。男性组和女性组PRP中血小板浓度和白细胞浓度比较差异均无统计学意义(P>0.05)。PRP中PDGF、TGF-β、VEGF浓度分别为(698.15±64.48)、(681.36±65.90)、(1071.55±106.04)ng/mL,是全血的(5.67±1.18)、(6.99±0.61)、(5.74±0.83)倍。PRP中PDGF浓度(r=0.832,P=0.020)、TGF-β浓度(r=0.835,P=0.019)、VEGF浓度(r=0.824,P=0.023)均与PRP中血小板浓度成正相关。结论 PRP制备套装可以稳定地制备出富含高浓度血小板、白细胞和生长因子的PRP。     

兔脂肪来源干细胞分离培养及富血小板血浆对其增殖的影响

目的 探讨自体富血小板血浆(PRP)对脂肪来源T细胞(ADSCs)增殖的影响.方法 选取新西兰大白兔的颈背部区脂肪垫,采用贴壁法及Ⅰ型胶原酶消化法行体外培养兔脂肪来源干细胞,观察其形态特征;取P3代细胞进行成脂、成骨诱导分化,以油红0、茜素红染色鉴定;抽取兔心脏血,经离心处理后制备富血小板血浆,行血小板计数;采用CCK-8法检测不同作用时间(24、48、72 h)对脂肪来源干细胞增殖活动的影响.结果 兔脂肪来源干细胞原代及传代细胞呈长梭或多边彤贴壁生长;油红0及茜素红染色呈阳性;全血血小板计数为(313.0±137.5)×109/L,PRP血小板计数为(1267.0±760.2)×109/L,PRP血小板计数是全血的4.08倍;CCK-8法显示PRP组在24、48、72h较对照组增殖明显(P<0.01).结论 PRP对体外培养的兔脂肪来源干细胞的增殖有明显的促进作用.     

腱骨愈合过程中富血小板血浆对细胞增殖与胞浆内钙离子浓度的影响

目的 研究富血小板血浆(PRP)在腱骨愈合过程中对肌腱细胞、成骨细胞的增殖情况及胞浆内钙离子浓度的影响.方法 用Transwell小室建立共培养模型,用CCK-8试剂盒检测细胞增殖能力,用激光共聚焦显微镜检测经钙离子荧光探针fluo-3/AM染色的胞浆内钙离子浓度的变化.单独培养成骨细胞组、肌腱细胞组,单独培养成骨细胞组、肌腱细胞组并各自加入PRP(加入小室上层),分别以成骨细胞和肌腱细胞为待测细胞建立共培养体系但不加入PRP组,分别以成骨细胞和肌腱细胞为待测细胞建立共培养体系并且加入PRP组. 结果 2种细胞共培养且未加入PRP两组生长速度和荧光强度均为最低,差异有统计学意义(P＜0.05),2种细胞单独培养且未加入PRP的2种细胞生长速度和荧光强度均为居中,差异有统计学意义(P＜0.05),加入PRP的各组生长速度和荧光强度均为最高,且同种细胞间比较差异无统计学意义(P＞0.05).结论 PRP可以去除2种细胞共培养时彼此的抑制效应,并提高细胞的增殖能力至同样高的水平,且胞浆内钙离子浓度也随之升高.     

自体血小板分离在心血管手术中的应用

体外循环(CPB)期间,由于血小板激活和α-颗粒释放,导致血小板减少和功能下降。CPB前,利用自体血小板分离技术可将部分血小板从患者全血中分离出来制成富血小板血浆(platelet-richplasma,PRP),PRP可在术后回输,以达到血小板数量和功能的双重保护,另一方面可将PRP制备成自体血小板胶(autologousplateletgel,APG),APG中含有丰富的生长因子,并且具有足够的抗张强度和粘性,因此可起到术中止血、封闭伤口、促进胸骨及伤口愈合的作用。     

富血小板血浆治疗下肢慢性难愈合伤口47例随访研究

目的 探讨富血小板血浆(platelet-rich plasma,PRP)对下肢慢性难愈合伤口的修复作用. 方法 2007年5月-2007年11月,采用PRP注射治疗下肢慢性难愈合伤口47例.男41例,女6例;年龄15～68岁,平均43.2岁.原发疾病:胫腓骨骨折20例,跟骨骨折4例,跖骨骨折1例,下肢多发开放性骨折3例,胫骨骨髓炎10例,股骨骨髓炎1例,足踝部软组织损伤4例,截肢术后感染2例,足部矫形术后感染及跟腱修补术后感染各1例.外院治疗后2～4个月创口未愈合转入合并骨折未愈合23例,细菌培养结果 阳性38例.患者予2次清创加自体PRP伤口内注射,每次间隔2个月. 结果 患者均于首次注射PRP后获随访,随访时间4个月.首次注射PRP2个月后,34例伤口明显缩小,坏死组织及脓苔清除,组织色泽健康,血供良好,外露骨或肌肉组织被新牛肉芽组织覆盖.4个月随访时,无肌肉和骨组织外露患者,创面覆盖率79.3%4±18.O%,总治愈率29.8%.治疗前创口体积(11.8±5.6)mL,治疗后为(2.5±2.7)mL,创口体积缩小(9.3±4.9)mL,治疗前后创口体积比较差异有统计学意义(P＜0.05).术前23例合并骨折未愈合者,随访4个月时骨折完全愈合9例,骨痂生长明显增多12例,无明显改变2例,均无骨髓炎征象加重.细菌培养阳性结果 15例. 结论 PRP能有效促进软组织缺损修复,加速下肢慢性难愈合伤口愈合.     

胸壁窦道病人应用富血小板血浆凝胶联合负压封闭引流的临床疗效观察

目的 探讨胸壁窦道病人应用富血小板血浆凝胶(APG)联合负压封闭引流(VSD)的临床疗效。方法 2019年10月～2021年3月收治的胸壁窦道病人89例,采用随机数表法分为观察组(45例)及对照组(44例),对照组采用VSD治疗,观察组采用APG联合VSD治疗,比较两组胸壁窦道愈合情况。结果 观察组和对照组总有效率分别为97.78%和86.36%,两组比较差异有统计学意义(P<0.05);观察组窦道封闭时间、创面愈合时间、住院时间分别为(15.26±2.33)天、(25.19±3.54)天和(41.26±5.33)天,短于对照组的(27.26±3.05)天、(43.26±5.17)天和(58.64±6.11)天,差异有统计学意义(P<0.05),观察组二次修复手术率、窦道复发率均为0,低于对照组的11.36%和13.64%,差异有统计学意义(P<0.05);治疗后2周观察组C反应蛋白(CRP)、白细胞计数(WBC)、肿瘤坏死因子(TNF)-α、疼痛评分(VAS)分别为(20.36±3.41)mg/L、(5.23±0.64)×10⁹、(15.26±...     

富血小板血浆对大鼠许旺细胞生物学行为的影响

目的 观察不同浓度的富血小板血浆(PRP)对体外培养的许旺细胞(SCs)增殖、分泌功能及迁移的影响,探讨其促进周围神经再生的可能作用机制. 方法 从SD大鼠心脏穿刺取血,利用二次离心法制备PRP,对全血和PRP中血小板计数和血小板源性生长因子BB (PDGF-BB)和转化生长因子(TGF-β1)浓度测定;取3～5d龄的大鼠坐骨神经培养纯化SCs,将P1细胞分为实验组与对照组分别进行处理,实验组以含40.0％、20.0％、10.0％、5.0％和2.5％ PRP的条件培养液干预,并设立空白对照组.于干预不同时间点采用CCK-8法测定SCs增殖活性情况,用实时荧光定量PCR方法检测细胞神经生长因子(NGF)和胶质细胞源神经营养因子(GDNF) mRNA表达的变化,ELISA检测SCs分泌NGF和GDNF的水平,Transwell小室检测各组SCs的迁移能力. 结果 PRP血小板回收率达65％,PDGF-BB和TGF-β1浓度明显高于血清(P＜0.01);与空白对照组相比,低于20 0％浓度的PRP呈浓度依赖性促进SCs增殖和迁移,而40.0％浓度组细胞增殖和迁移受到抑制;SCs分泌的NGF和GDNF和其mRNA表达均较对照组明显增加,同样在低于20.0％浓度的范围内呈现量效关系,40.0％浓度组则显示抑制作用.结论 PRP在适当浓度范围可以促进SCs的分裂增殖,合成分泌NGF和GDNF以及迁移的能力,具有潜在的促周围神经再生的作用.     

血小板源性生长因子BB对大鼠肌腱愈合和肌腱粘连的影响

目的 了解血小板源性生长因子BB(PDGF-BB)基因转染大鼠肌腱细胞对肌腱愈合及肌腱粘连的影响. 方法 将90只SD大鼠制成跟腱损伤模型,按随机数字表法分为3组,每组30只:实验组,肌腱断端注射20μL转染PDGF-BB基因的大鼠肌腱细胞(1×108个/mL);对照组,肌腱断端注射20μL未行转染的大鼠肌腱细胞(1×108个/mL);空白对照组,不做任何处理.6-0丝线行改良Kessler法缝合跟腱,管型石膏固定1周.通过基因测序及RT-PCR鉴定转染PDGF-BB基因的大鼠肌腱细胞.分别于术后3 d和1、2、4、8周取各组大鼠肌腱组织样本,行大体、组织学观察以及生物力学检测,对比各组肌腱粘连度、组织中Fb数量与胶原纤维含量、肌腱最大抗拉力及最大滑动距离、组织中PDGF-BB的浓度.对数据行t检验. 结果(1)转染的肌腱细胞经RT-PCR以及基因测序证实在体外稳定表达PDGF-BB mRNA.(2)各组大鼠术后3 d肌腱均出现较明显肿胀及炎性细胞浸润,实验组改变较其他组明显轻微;随后各组情况均逐渐好转.术后4、8周肌腱粘连度分级组间比较未见明显差异.(3)实验组Fb数在术后2、4、8周显著低于对照组和空白对照组(t值分别为2.94、4.26、5.76和4.00、3.83、6.12,P＜0.05或P＜0.01).(4)实验组术后4周胶原纤维含量为(43±6)%,较对照组[(55±8)%]与空白对照组[(61±8)%]显著下降(t值分别为2.94和4.41,P＜0.05或P＜0.01).(5)术后4、8周实验组肌腱最大滑动距离为(3.25±0.33)、(3.65±0.21)mm,显著高于对照组的(2.29±0.40)、(2.21±0.37)mm和空白对照组的(2.01±0.23)、(1.89±0.24)mm(t值分别为4.53、8.29和7.55、13.52,P值均小于0.01),但其肌腱最大抗拉力与另2组比较差异无统计学意义(t值分别为0.41、0.41和0.77、0.72,P值均大于0.05).(6)术后3 d和2、4周,实验组肌腱组织中PDGF-BB浓度为(12.95±1.36)、(8.32±0.94)、(9.10±1.06)ng/mL,均显著高于对照组的(1.13±0.21)、(2.07±0.48)、(3.85±0.39)ng/mL(t值分别为21.04、14.50、11.39,P值均小于0.01). 结论 转染PDGF-BB基因肌腱细胞有促进肌腱内源性愈合、减轻肌腱粘连的作用.     

富含血小板血浆对小鼠触须毛乳头细胞活性和毛囊重建的影响

目的 探讨富含血小板血浆(platelet-rich plasma,PRP)对小鼠触须毛乳头细胞(dermal papilla cells,DPCs)活性和毛囊重建的影响.方法 将采用2步离心法制备的PRP与采用显微解剖法获得的DPCs共培养,显微镜下观察细胞的形态变化,通过MTT法检测第4和第8代DPCs细胞活性.将新鲜分离的C57BL/6J小鼠背部表皮细胞与第4代的DPCs混合制成含有不同终浓度PRP的细胞悬液并移植至裸鼠体内,观察移植区域毛发形成的时间,4周后处死裸鼠,HE染色计算各组毛囊数量.结果 通过2步离心法获得的PRP中含有的血小板数量约为全血的8.3倍,与未加PRP的对照组比较,终浓度为5％和10％ PRP组不但可促进前6代DPCs的聚集性生长,还可促进前8代DPCs的增殖.在裸鼠体内实验中,10％ PRP组长出毛发的最早时间和形成毛囊的数量分别为(18±1)d和(344±27)根,对照组为(20±1)d和(288±35)根,差异有统计学意义(P＜0.05).结论 PRP既能够增强DPCs的活性,也有助于毛囊的重建.     

The contribution of platelet-derived growth factor, transforming growth factor-beta1, and insulin-like growth factor-I in platelet-rich plasma to the proliferation of osteoblast-like cells.

The aim of this study was to investigate the effect of platelet-rich plasma (PRP) on the proliferation of osteoblast-like cells in vitro. PRP was prepared using a centrifuge; the number of platelets (n = 32) and the levels of platelet-derived growth factor-AB (PDGF-AB), transforming growth factor-beta1 (TGF-beta1), and insulin-like growth factor-I (IGF-I) were measured (n = 16). For the proliferation assay, SaOS-2 was cultured in the presence of platelet-poor plasma (PPP), whole blood, or PRP. The cell number was counted after 36 and 72 hours. To investigate the effect of each growth factor, the cells were cultured with PRP in the absence or presence of neutralizing antibodies, and counted as described. The mean platelet count of PRP was 1546.36 +/- 382.25 x 10(3)/microL, and the mean levels of PDGF-AB, TGF-beta1 and IGF-I were 0.271 +/- 0.043, 0.190 +/- 0.039, and 0.110 +/- 0.039 ng/1500 x 10(3) platelets, respectively. Cell proliferation was enhanced in all PRP groups in a dose-dependent manner, and all neutralizing antibodies significantly suppressed proliferation compared with the PRP group, lacking antibody, at 36 hours. However, at 72 hours, the neutralizing antibodies of PDGF and TGF-beta1, but not IGF-I, significantly suppressed proliferation. These results show the beneficial abilities of PRP in the proliferation of osteoblast-like cells from the standpoint of growth factors, including the contribution of each factor.     

Autologous platelet gel in coronary artery bypass grafting: effects on surgical wound healing

Stimulating the body's natural healing at the cellular level can be achieved through the application of growth factors located within platelets. Once combined with a mixture of calcium and thrombin, this substance, now referred to as autologous platelet gel (APG), can be applied to surgical wound sites for patients undergoing cardiac surgery. The purpose of this study was to examine the effects of APG on surgical site infection, post-operative pain, blood loss, and bruising. After 30 mL platelet-rich plasma (PRP) was processed, 10 mL PRP was distributed on the sternum after re-approximation and 7 mL PRP before skin closure. Ten milliliters PRP was used on the endoscopic leg harvest (EVH) site. The remaining 3 mL was sent to the laboratory for hematologic testing. Both the control (CTR) and treatment (TRT) groups were well matched, with the exception of ejection fraction and pre-operative platelet count, which was significantly higher in the TRT group. Average platelet count yield was 4.2 +/- 0.5 x 103/mcL, white blood count (WBC) yielded 1.9 +/- 0.7 x 103/mcL, and fibrinogen yielded 1.2 +/- 0.2 mg/dL above baseline. There were no deep or superficial sternal infections. However, one patient from each group did experience a leg infection at the EVH site, which occurred after hospital discharge. More patients in the TRT group experienced less pain on postoperative day (POD) 1 and at the post-operative office follow-up. Blood loss and bruising was less in the TRT group on POD 2; however, there was no statistical significance. The application of APG seems to confer beneficial effects on pain, blood loss, and bruising. However, further studies with a greater sample size are needed to power significant differences.     

Sodium nitroprusside during coronary artery bypass grafting: evidence for an antiinflammatory action.

BACKGROUND: It was the aim of the present study to investigate whether a nitric oxide donor can reduce systemic inflammation and the cardiac inflammatory response during coronary artery bypass grafting with cardiopulmonary bypass. METHODS: Patients undergoing elective coronary artery bypass grafting (n = 22) were randomly assigned to treatment with either sodium nitroprusside (0.5 microg x kg(-1) x min(-1)) or placebo (controls), both for the first 20 minutes of reperfusion. Interleukin-6 and interleukin-8 levels, the adhesion molecules CD41 and CD62 on platelets and CD41 on monocytes and PMN (as markers for coaggregate formation), CD11b on monocytes and PMN, as well as platelet and leukocyte counts were determined in radial artery and coronary sinus blood before cardiopulmonary bypass and during reperfusion (1, 5, 10, 25, and 35 minutes). RESULTS: A reduction of systemic interleukin-6 levels (15.4+/-3.5 pg/mL, 36.7+/-5.9 pg/mL, and 46.8+/-8.0 pg/mL versus 33.4+/-7.7 pg/mL, 76.7+/-13.2 pg/mL, and 106.0+/-26.5 pg/mL, respectively, at 1, 25, and 35 minutes of reperfusion) and interleukin-8 (29.6+/-4.5 pg/mL versus 54.0+/-9.4, pg/mL, resp., at 35 minutes of reperfusion) resulted from treatment with sodium nitroprusside. No intracardiac production of interleukin-8 in sodium nitroprusside-treated patients (-1.1+/-0.4 pg/mL and -2.8+/-2.2 pg/mL, resp., for the coronary sinus-radial artery difference at 5 and 25 minutes of reperfusion) was observed, whereas cardiac production of interleukin-8 was present in controls (2.5+/-1.5 pg/mL and 5.5+/-2.8 pg/mL, resp.). Retention of platelet/leukocyte coaggregates occurred during coronary passage in controls (coronary sinus-radial artery difference for CD41-positive monocytes at 1 and 10 minutes of reperfusion, -16.3%+/-8.5% and -8.8%+/-2.6%, resp.). This was reduced in sodium nitroprusside-treated patients (with 5.8%+/-5.2% and 0.0%+/-3.2%). Retention of platelets in controls (ratio of coronary sinus to radial artery platelet count at 5 and 10 minutes of reperfusion, 88%+/-6% and 91%+/-5%) was compared to washout in treated patients (108%+/-6% and 113%+/-7%). CONCLUSIONS: In patients undergoing routine coronary artery bypass grafting, administration of sodium nitroprusside during early reperfusion alleviates systemic inflammation and the cardiac inflammatory response.     

Better preservation of endothelial function and decreased activation of the fetal renin-angiotensin pathway with the use of pulsatile flow during experimental fetal bypass

OBJECTIVE: Pulsatile flow was shown to overcome the progressive rise in peripheral and placental vascular resistances observed during steady-flow bypass, this rise being counteracted by inhibition of nitric oxide synthase. This study quantifies the release of endothelial vasoactive substances during a 60-minute in utero model of fetal bypass. METHODS: Fetuses were randomly allocated into 1 of 2 groups (steady flow, n = 8, or pulsatile flow, n = 13) and subjected to bypass through central cannulation and perfusion with either a centrifugal or pulsatile (125 beats x min(-1)) blood pump. RESULTS: Lactate concentration was high, starting at fetal exteriorization and increasing during fetal preparation in the 2 groups. Once bypass was established, the rise was significant only in the steady-flow group. Plasma nitric oxide metabolites, similar before bypass, reached higher levels during pulsatile flow at the end of bypass (99+/-9 vs. 82+/-23 micromol x L(-1); P =.037). Levels of urinary nitric oxide metabolites were significantly higher in the pulsatile-flow than in the steady-flow group (764+/-143 vs. 508+/-240 micromol x L(-1); P =.005). Plasma cyclic guanosine monophosphate levels increased after 30 minutes of bypass in the pulsatile-flow group (25+/-18 vs. 12+/-8 pmol x mL(-1); P =.004), and urinary cyclic guanosine monophosphate excretion was higher in the pulsatile-flow group (517+/-450 vs. 118+/-78 pmol x mL(-1); P =.024). Plasma endothelin-1 levels increased in the 2 groups and were higher in the steady-flow group at 30 minutes (27+/-5 vs. 23+/-2 pg x mL(-1); P =.04) and 60 minutes of bypass (39+/-7 vs 32 +/- 6 pg x mL(-1); P =.04). Plasma renin concentration increased significantly during bypass only in the steady-flow group (26+/-10 vs. 57+/-42 in ng A1 x mL(-1) x h(-1); P =.04). CONCLUSIONS: Improved placental and peripheral perfusion during fetal pulsatile-flow bypass may be mediated by preservation of fetal/maternal endothelial nitric oxide biosynthetic mechanisms and/or decreased activation of the fetal renin-angiotensin pathway.     

Transforming growth factor-beta1 increases albumin permeability of isolated rat glomeruli via hydroxyl radicals

BACKGROUND: Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine. Glomerular cells and tubular epithelial cells secrete and respond to TGF-beta1. A close association between elevated levels of TGF-beta1 and the development of glomerulonephritis, glomerulosclerosis, and tubular hypertrophy has been documented. The role of TGF-beta1 in proteinuria is not well understood. METHODS: Isolated rat glomeruli were incubated in medium alone or with TGF-beta1 (1 to 10 ng/mL) and TGF-beta1 + 200 U/mL of superoxide dismutase (SOD) or 1 mmol/L of dimethylthiourea (DMTU) scavengers of superoxide and hydroxyl radicals, respectively, for up to 60 minutes at 37 degrees C. Glomerular albumin permeability (Palb) was calculated from the volumetric response of glomeruli to an oncotic gradient using videomicroscopy. RESULTS: One or 2.5 ng/mL of TGF-beta1 had no effect on Palb (0.18 +/- 0.08, N = 17; 0.18 +/- 0. 079, N = 20 vs. control 0.00 +/- 0.06, N = 25), whereas 5 or 10 ng/mL of TGF-beta1 caused a significant increase in Palb (0.31 +/- 0. 09, N = 20; 0.33 +/- 0.06, N = 23) within 15 minutes. The effect of 10 ng/mL of TGF-beta1 on Palb increased further after 30, 45, or 60 minutes of incubation (0.43 +/- 0.06, N = 24; 0.53 +/- 0.06, N = 25; 0.74 +/- 0.075, N = 21). The TGF-beta1-induced increase in Palb (0. 75 +/- 0.065, N = 15) was blocked by SOD (0.07 +/- 0.14 N = 15) or by DMTU (0.04 +/- 0.13, N = 15). Incubation of glomeruli with the carrier medium (4N HCl) in which TGF-beta1 is dissolved and SOD or DMTU alone did not affect Palb. CONCLUSION: Elevated levels of TGF-beta1 derived from glomerular or extraglomerular sources are capable of increasing glomerular Palb via superoxide and hydroxyl radicals and may lead to proteinuria in vivo.     

Circulating VEGF and TGF-beta1 in children with idiopathic nephrotic syndrome

RATIONALE: The implications of vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-beta) on the development of proteinuria were studied by measuring the mRNA and protein levels of VEGF and TGF-beta1 in 43 children with primary nephrotic syndrome. METHODS: Twenty-seven patients were in the active nephrotic phase at the time of sampling (Group 1), and 16 were in remission (Group 2). In Group 1, 16 were steroid-responders (Group 1a) and 11 were nonresponders (Group 1b). Minimal change lesion (MCL) in 11 patients and focal segmental glomerulosclerosis (FSGS) in 8 were confirmed by renal biopsy. The mRNA expressions of peripheral blood lymphocytes and the plasma levels of proteins were measured by semi-quantitative RT-PCR and ELISA, respectively. RESULTS: Plasma VEGF concentration was higher in Group 1 (204+/-137 pg/mL) than Group 2 (91+/-72 pg/mL) (P=0.002). However, there was no significant difference either between Group 1a (184+/-146 pg/mL) and Group 1b (258+/-134 pg/mL) or between patients with FSGS (330+/-122 pg/mL) and those with MCL (146+/-112 pg/mL). The VEGF mRNA expression showed changes similar to VEGF protein expression, and there was no statistical significance. Plasma levels and mRNA expressions of TGF-beta1 were similar in all groups. CONCLUSIONS: These results suggest that circulating VEGF is associated with proteinuria both in steroid-responsive and steroid-resistant primary nephrotic syndrome in children.     

Decompensation characterized by decreased perfusion of the heart and brain during hemorrhagic shock: role of endothelin-1

BACKGROUND: Endothelin-1 (ET-1), a 21-amino-acid peptide produced by vascular endothelium, is a potent vasoconstrictor and a component of local regulation of vascular tone through its effect on underlying vascular smooth muscle. Hemorrhagic shock (HS) is characterized by compensatory regional vasoconstriction to decrease peripheral tissue perfusion and to maintain core organ perfusion. Decompensation occurs with prolonged duration of HS. In the present study, we hypothesized that systemic and vital organ tissue ET-1 concentrations would correlate with changes in systemic and vital organ perfusion associated with compensatory and decompensatory states of HS. METHODS: After surgical instrumentation, HS was induced in male Sprague-Dawley rats by withdrawing blood via femoral artery to a mean arterial pressure of 35 to 40 mm Hg that was maintained for either 30 minutes or 90 minutes in separate groups of male Sprague-Dawley rats. Systemic hemodynamics and regional blood flow were measured using a radioactive microsphere technique. In separate groups of animals, sham, 30 minutes of HS, or 90 minutes of HS, plasma and tissue concentrations of ET-1 were determined using a radioimmunoassay technique. RESULTS: HS maintained for 90 minutes was associated with increased arterial base deficit from 3.6 +/- 0.53 mEq/L to 13 +/- 0.37 mEq/L, decreased cardiac output from 79 +/- 18 mL/min to 18 +/- 5 mL/min, and increased systemic vascular resistance from 1,004 +/- 102 mm Hg/L. min to 2,392 +/- 447 mm. Hg/L min as compared with baseline values. With 90 minutes of HS as compared with 30 minutes of HS, perfusion was significantly decreased in brain (72 +/- 11 vs. 29 +/- 6 mL/min. 100 g tissue) and heart (483 +/- 30 vs. 173 +/- 38 mL/min. 100 g tissue) and kidney perfusion was decreased (from 114 +/- 28 mL/min/100 g tissue to 29 +/- 2 mL/min. 100 g tissue), and ET-1 concentration was increased significantly in brain (cerebral cortex, 89 +/- 14 pg/100 g tissue to 144 +/- 19 pg/100 g tissue; midbrain, 172 +/- 15 pg/100 g tissue to 211 +/- 10 pg/100 g tissue), heart (left ventricle, 312 +/- 11 pg/100 g tissue to 360 +/- 14 pg/100 g tissue), kidney (medulla, 857 +/- 61 pg/100 g tissue to 1,277 +/- 41 pg/100 g tissue), and plasma (5.31 +/- 0.6 pg/100 g tissue to 21.26 +/- 2.9 pg/mL). CONCLUSION: Decreased vital organ and peripheral tissue perfusion, a primary decompensation effect of HS, was apparent with 90 minutes of HS but not with 30 minutes, and was associated with increased vital organ tissue and plasma ET-1 concentrations. These data suggest a role for ET-1 in control mechanisms of progressive vasoconstriction that occurs with prolonged duration of HS.     

Low TGF-beta1 serum levels are a risk factor for atherosclerosis disease in ESRD patients.

BACKGROUND: It is thought that transforming growth factor-beta1 (TGF-beta1) might be a key inhibitor of atherogenesis in non-uremic patients. We evaluated the intra- and post-dialytic serum levels of TGF-beta1 in uremic patients to assess if TGF-beta1 is an independent risk factor for cardiovascular diseases, and if any correlation exists between TGF-beta1 and any yet known atherosclerotic risk factors. METHODS: We studied 155 patients who were on regular hemodialysis, with or without clinically significant atherosclerotic vascular disease. Forty-one apparently healthy people were enrolled as a control group. TGF-beta1 was evaluated during the midweek dialysis session, at times 0, 30, and 120 minues, at the end of the session, and 3 hours after the session's end. All hitherto known atherosclerotic risk factors also were evaluated. The investigation was performed over a 24-month follow-up. RESULTS: TGF-beta1 values (mean +/- SD) in dialysis patients were 26.64 +/- 7.0 ng/mL (N=155) compared with 42.31 +/- 6.0 ng/mL in the control group (N=41, P < 0.0001). A weak inverse correlation emerged between TGF-beta1 and age (r=-0.28), TGF-beta1 and lipoprotein(a) [Lp(a); r=-0.35], TGF-beta1 and C-reactive protein (CRP; r=-0.27), and TGF-beta1 and plasminogen activator inhibitor-1 (PAI-1; r=-0.41). TGF-beta1 also correlated with albumin (r=0.31). In the coronary heart disease (CHD) group (N=32) the TGF-beta1 was 26.2 +/- 4.9 ng/mL; in the cerebrovascular disease (CVD) group (N=8) it was 26.7 +/- 3.7 ng/mL and in the peripheral vascular disease (PVD) group (N=9) it was 25.4 +/- 1.7 ng/mL. In dialysis patients with no cardiovascular disease (N=80) TGF-beta1 was 35.1 +/- 6.8 ng/mL (P < 0.0001 vs. CHD, CVD and PVD patients). TGF-beta1 was significantly lower among those patients with triple coronary vessel disease than with the other CHD patients. The Cox analysis demonstrated that a 1 ng/mL reduction in TGF-beta1 concentration was associated with a 9% increase in the relative risk of a cardiovascular event. CONCLUSIONS: TGF-beta1 was significantly reduced in hemodialysis patients, in particular in those with severe cardiovascular disease. Baseline TGF-beta1, diabetes mellitus and serum albumin levels proved to be the only independent contributors to atherosclerotic risk in dialysis patients.     

Plasma transforming growth factor-β1 levels in patients with erectile dysfunction

Aim: To evaluate the plasma TGF-β1 level in erectile dysfunction (ED) patients of various causes. Methods: Sixty-two patients with ED and 26 potent men were subjected to the study. Based on multidisciplinary work-ups, including medical history, physical examinations, blood tests with lipid profile and hormones, penile duplex Doppler ultrasonogram and neurophysiological tests, causes for ED were classified as psychogenic (n=15), neurogenic (n=16) and vasculogenic (n=31). The plasma TGF-β1 level was measured by the ELISA method. Results: The plasma TGF-β1 level was significantly increased in the ED group (6.7 ± 4.9 ng/mL), compared to the control (4.0±2.1 ng/mL) (P <0.01). In the ED groups, there was a significant increase in the vasculogenic group (9.0 ± 5.5 ng/mL), compared to the psychogenic (3.8 ± 1.8 ng/mL) and neurogenic groups (4.8 ± 3.2 ng/mL) (P<0.01). Of the vascular risk factors, both the smoking (7.5 ± 4.7 ng/mL) and dyslipidemia groups (7.4 ± 4.4 ng/mL) showed significantly increased