Regulation of matrix contraction in chronic venous disease.
Regulation of matrix contraction in chronic venous disease.
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慢性静脉疾病中基质收缩的调节。
DOI：
10.1016/j.ejvs.2009.05.012
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发表时间：
2009
期刊：
影响因子：
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通讯作者：
Durán,WN
中科院分区：
文献类型：
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作者：
Pappas,PJ;Lal,BK;Ohara,N;Saito,S;Zapiach,L;Durán,WN
目的转化生长因子-β-1在静脉性溃疡愈合中的作用及其调控真皮成纤维细胞功能的信号转导机制尚不清楚。为了阐明这些过程，我们假设转化生长因子-β-1通过增加慢性静脉功能不全引起的基质收缩而促进创面愈合。方法从不同程度的慢性静脉功能不全患者的小牛活检组织中分离出成纤维细胞，在等长条件下种植于200μL胶原凝胶中。对照组为新生儿包皮成纤维细胞(HS68)、非CVI患者(NC)和CVI患者的同侧正常大腿(LT)。13例CVI患者(2级5例，4级5例，6级3例)和2例非CVI对照组(NC，n=2)纳入研究。所有实验条件均通过剂量-反应和时间-过程实验确定。加入/或不加0.1 ng/mlTGFAPK1和加/不加50μMPD98059(MEK及其下游MAPK抑制剂)培养凝胶。另外，将活性RAS(pCMV-RAS)或空载体(pCMV-β)分别与0.1 ng/mlTGF-β1和50μmPD98059共同导入患者成纤维细胞。胶原凝胶在4天后释放，并通过图像分析测量面积来确定收缩百分比。Western blotting分析α-平滑肌肌动蛋白(α-SMA)和ERK1/2MAPK(磷酸化和总)蛋白表达水平的差异。与未受刺激的对照组相比，抑制丝裂原活化蛋白激酶和/或用转化生长因子-β1刺激可增加LC凝胶的收缩。激动剂诱导的凝胶收缩与CVI疾病严重程度相关。在有或无α-β1刺激的情况下，丝裂原活化蛋白激酶被抑制的LC成纤维细胞的SMA蛋白表达增加，并与凝胶收缩程度相关。PCMV-RAS(ERK-1/2激活剂)可抑制凝胶收缩，但这种抑制作用不能被转化生长因子-β-1逆转。空载体pCMV-β对凝胶收缩无影响。结论β-1可通过上调α-SMA的表达而促进凝胶收缩，从而促进凝胶收缩。抑制MAPK进一步增加凝胶收缩，而激活ERK-1/2的RAS则抑制转化生长因子-β1诱导的凝胶收缩。这些反应与CEAP严重程度的增加有关。因此，CVI成纤维细胞介导的凝胶收缩是通过ERK-1/2MAPK和转化生长因子-β1信号通路之间的串扰来调节的。这些数据确定了潜在的临床相关治疗分子靶点，这些靶点可以增强基质收缩，从而改善静脉溃疡伤口的愈合。
OBJECTIVEThe role of TGF-β1in venous ulcer healing and the signalling cascades regulating dermal fibroblast function are poorly understood. To elucidate these processes, we hypothesized that TGF-β1facilitates wound healing by increasing chronic venous insufficiency (CVI) induced matrix contraction via intracellular cross-talk between TGF-β1and the ERK-1/2 MAP kinase signalling cascades.METHODSFibroblasts isolated from calf biopsies (LC) of patients with different severity of CVI (CEAP, Clinical Etiological Anatomical Pathological classes) were seeded into 200μl collagen gels under isometric conditions. Fibroblasts from neonatal foreskins (HS68), non-CVI patients (NC), and the ipsilateral normal thigh of each CVI patient (LT) served as controls. Thirteen patients with CVI (class 2, n=5; class 4, n=5; class 6, n=3) and 2 non-CVI controls (NC, n=2) were included in the study. All experimental conditions were determined by dose–response and time-course experiments. Gels were cultured with/without 0.1ng/ml TGF-β1and with/without 50μM PD98059 (MEK and downstream-MAPK inhibitor). Additional patient fibroblasts were transfected with constitutively active Ras (pCMV-Ras) or an empty vector (pCMV-β) with/without 0.1ng/ml TGF-β1and with/without 50μm PD98059. The collagen gels were released after 4 days and the percent contraction was determined by area measurements using image analysis. Differences in α-smooth muscle actin (α-SMA) and ERK-1/2 MAPK (phosphorylated and total) protein levels were analyzed with western blotting.RESULTSGels seeded with CVI fibroblasts contracted more than HS68, NC and LT fibroblasts. Inhibition of MAPK and/or stimulation with TGF-β1increased the contraction of LC gels compared to unstimulated controls. Agonist induced gel contraction correlated with CVI disease severity. α-SMA protein expression in LC fibroblasts increased with MAPK inhibition with/without TGF-β1stimulation, and correlated with the degree of gel contraction. Transfection with pCMV-Ras (activator of ERK-1/2) inhibited gel contraction; this inhibition was not reversed by addition of TGF-β1. Transfection with the pCMV-β empty vector had no effect on gel contraction.CONCLUSIONSTGF-β1 stimulation of CVI patient fibroblasts grown in 3D collagen gels results in conversion to a contractile phenotype through upregulation of α-SMA, and in enhanced gel contraction. Inhibition of MAPK further increases gel contraction, while Ras activation of ERK-1/2 inhibits TGF-β1-induced gel contraction. These responses correlate with increasing CEAP severity. CVI fibroblast mediated gel contraction is therefore regulated through cross-talk between the ERK-1/2 MAPK and TGF-β1signalling cascades. These data identify potentially clinically relevant therapeutic molecular targets that could enhance matrix contraction and thereby improve venous ulcer wound healing.