Differential response of human cardiac fibroblasts to angiotensin I and angiotensin II

The vasoactive peptide angiotensin II (Ang II) has been implicated as a mediator of myocardial fibrosis. We carried out a comparative investigation of the effects of Ang II and its precursor Ang I on collagen metabolism and proliferation in cultured human cardiac fibroblasts. Cardiac fibroblasts responded to both Ang I and Ang II with concentration-dependent increases in collagen synthesis but no proliferation. The stimulatory effect of Ang II was abolished by the AT(1) receptor antagonist losartan but not the AT(2) receptor antagonist PD123319. The response to Ang I was not affected by either antagonist, nor by the angiotensin-converting enzyme (ACE) inhibitor captopril. In conclusion, Both Ang I and Ang II stimulate collagen synthesis of human cardiac fibroblasts, the effect of Ang II occurring via the AT(1) receptor whilst Ang I appears to exert a direct effect through non-Ang II-dependent mechanisms. These results suggest distinct roles for angiotensin peptides in the development of cardiac fibrosis.     

PPARbeta/delta activation inhibits angiotensin II-induced collagen type I expression in rat cardiac fibroblasts

Peroxisome proliferator-activated receptors (PPARalpha, beta/delta and gamma) are nuclear receptors and PPARgamma activation was previously reported to inhibit collagen expression in the heart, but whether PPARbeta/delta also regulates collagen expression in the heart remains unclear. In this study, we investigated the effect of PPARbeta/delta activation on angiotensin II (Ang II)-induced collagen type I expression in adult rat cardiac fibroblasts. The results showed that PPARbeta/delta was expressed at the moderate level in cardiac fibroblasts. GW501516, a selective PPARbeta/delta agonist, depressed Ang II-stimulated collagen type I expression and collagen synthesis in cardiac fibroblasts in a concentration-dependent manner. Furthermore, these inhibitory effects of GW501516 were completely reversed by the knockdown of PPARbeta/delta via RNA interference. In summary, we find that PPARbeta/delta is present in cardiac fibroblasts and PPARbeta/delta activation inhibits Ang II-induced collagen type I expression at least in part via decreasing collagen synthesis. PPARbeta/delta may be a promising therapeutic target for myocardial fibrosis.     

血管紧张素Ⅱ诱导心肌成纤维细胞表达上调基因的分离和鉴定

为了对成年大鼠心肌成纤维细胞（cardiac fibroblasts,CF）受血管紧张素Ⅱ（angiotensin Ⅱ,AngⅡ）刺激后上调基因表达谱进行筛选及分析,以受AngⅡ刺激CF为实验方,未刺激CF为驱动方,进行抑制消减杂交（suppression subtractive hybridization,SSH）,建立消减cDNA文库。经斑点杂交筛选文库后将表达变化显著的部分阳性克隆测序及同源性分析,共获得19个上调表达的基因,分别与细胞外基质、细胞周期、胞内信号转导、细胞骨架及细胞代谢等功能相关,并克隆到7个新的基因表达序列标签（expressed sequence tags,EST）。我们的数据证实了SSH可以有效地克隆成年大鼠CF受AngⅡ刺激后上调表达基因,对这些基因的研究将有助于阐明心肌重塑的分子机制。     

Cyclical mechanical stretch modulates expression of collagen I and collagen III by PKC and tyrosine kinase in cardiac fibroblasts

Mechanical load and chemical factors as stimuli for the different pattern of the extracellular matrix (ECM) could be responsible for cardiac dysfunction. Since fibroblasts can both synthesize and degrade ECM, ventricular fibroblasts from adult rat hearts underwent cyclical mechanical stretch (CMS; 0.33 Hz) by three different elongations (3%, 6%, 9%) and four different serum concentrations (0%, 0.5%, 5%, 10%) within 24 h. Expression of collagen I and III, as well as matrix metalloproteinase-2 (MMP-2), tissue inhibitor of MMP-2 (TIMP-2), and colligin were analyzed by RNase protection assay. In the absence of serum, 9% CMS increased the mRNA of collagen I by 1.70-fold and collagen III by 1.64-fold. This increase was prevented by the inhibition either of PKC or of tyrosine kinase but not of PKA. Inhibition of PKC or tyrosine kinase itself reduced the expression of collagen I and collagen III mRNA. The mRNA of MMP-2, TIMP-2, and colligin showed the same tendency by stretch. Combined with 10% serum, 6% CMS reduced the mRNA of collagen I (0.62-fold) and collagen III (0.79-fold). Inhibition of PKC or tyrosine kinase, but not of PKA, prevented the reduction of collagen I and collagen III mRNA in 10% serum. The results show that the response of fibroblasts to CMS depends on the serum concentration. At least two signaling pathways are involved in the stretch-induced ECM regulation. Myocardial fibrosis due to ECM remodeling contributes to the dysfunction of the failing heart, which might be attributed to changes in hemodynamic loading.     

Spike formation by fibroblasts adhering to fibrillar collagen I gel

The fibrillar collagen I gel induced the formation of numerous dendritic cell-like protrusions (cell spikes) from the cell body, whereas monomeric collagen I induced typical cell spreading with filopodia and lamellipodia in skin fibroblasts. Peripheral, not central stress fibers appeared upon adhesion to fibrillar collagen gel, whereas both types of fibers were evident upon adhesion to monomeric collagen. Microtubules and vimentin filaments were elongated inside stress fibers along the terminal tip of cell spikes. Spike formation was totally inhibited by nocodazole and severely delayed by cytochalasin D. This suggests that cell spike formation is dependent on microtubules rather than on F-actin. We then investigated the intracellular signaling responsible for cytoskeleton organization to identify the key factor that induces cell spike morphology. During cell spike formation, FAK and CAS were activated. More CAS was activated in cells on fibrillar collagen gel than on the monomeric form, whereas FAK was activated to the same level on either. At 90 min of culture, Rac1 was activated in cells on monomeric collagen I, whereas Cdc42, Rac1 and RhoA were activated in cells on fibrillar collagen gel. These results suggest that microtubule organization via CAS and small GTPases is important for the cell spike formation that is involved in collagen gel contraction and in wound retraction in skin.     

Loss of PTEN expression by mouse fibroblasts results in lung fibrosis through a CCN2-dependent mechanism

Elevated adhesive signaling promotes fibrosis. Protein phosphatase and tensin homologue (PTEN) dephosphorylates focal adhesion kinase and suppresses the activation of Akt and hence suppresses adhesive signaling. Loss of PTEN expression is associated with lung fibrosis, but whether PTEN expression by type I collagen-expressing cells controls lung fibrosis is unclear. Here, we use mice expressing tamoxifen-dependent cre recombinase expressed under the control of a COL1A2 promoter/enhancer and mice harboring floxed-PTEN and/or floxed-CCN2 alleles to assess whether loss of PTEN expression by type I collagen producing cells results in lung fibrosis in a CCN2-dependent fashion. In vivo, loss of PTEN expression resulted in the overexpression of both collagen type I and the pro-adhesive matricellular protein connective tissue growth factor (CTGF/CCN2). However, α-smooth muscle actin expression was unaffected. Loss of CCN2 expression by lung fibroblasts rescues this phenotype; i.e.., mice deficient in both PTEN and CCN2 in collagen type I-expressing cells do not develop significant collagen deposition in the lung. PTEN expression by collagen type I-expressing cells controls collagen deposition; therapeutic strategies blocking CCN2 may be of benefit in blocking excessive collagen deposition in fibrosis.     

High glucose promotes the production of collagen types I and III by cardiac fibroblasts through a pathway dependent on extracellular-signal-regulated kinase 1/2

Hyperglycemia promotes fibrosis by increasing collagen synthesis, a process involving mitogen activated protein kinases (MAPKs). Several studies of diabetic cardiomyopathy have demonstrated an accumulation of collagen, including collagen types I and III, in the myocardium, leading to interstitial fibrosis, which is related to left-ventricular diastolic dysfunction. However, the mechanisms of hyperglycemia-induced collagen production in cardiac fibroblasts are poorly defined. In the present study, neonatal rat cardiac fibroblasts treated with high glucose (25 mM) were assessed by real time PCR and enzyme linked immunosorbent assay (ELISA) showed an increase in both the mRNA and protein level of collagen types I and III. These effects were not due to changes in osmotic pressure. Extracellular signal regulated kinase 1/2 (ERK1/2) was activated by high glucose level (25 mM), and treatment with PD98059 to block ERK phosphorylation significantly inhibited the mRNA and protein expression of collagen types I and III. These results suggest that high glucose accelerates the synthesis of collagen types I and III, and an ERK1/2 cascade in cardiac fibroblasts play an essential role in the control of collagen deposition by high glucose.     

血管紧张素Ⅱ受体阻断对心肌成纤维细胞信号转导相关基因表达谱的影响

为了研究血管紧张素Ⅱ（angiotensinⅡ,AngⅡ）受体在成年大鼠心肌成纤维细胞的信号转导机制,分离及培养成年Sprague-Dawley大鼠心肌成纤维细胞,采用免疫组化染色测定AngⅡ受体的蛋白表达。将细胞随机分为四组进行药物干预48h：AngⅡ组、AngⅡ＋losartan组、AngⅡ＋PD123319组和AngⅡ＋losartan＋PD123319组。抽提mRNA制备cDNA探针,与G蛋白耦联受体信号通路发现者基因芯片杂交,筛选表达差异的基因。发现血管紧张素Ⅱ 1型（angiotensinⅡ type1,AT1）受体被losartan阻断后,AngⅡ刺激的心肌成纤维细胞血管紧张素Ⅱ2型（angiotensinⅡ type2,AT2）受体蛋白高表达;34个基因表达差异在2倍以上,30个下调,4个上调,其最大改变不超过20倍;9条信号通路被活化：cAMP／PKA、Ca^2＋、PKC、PLC、MAPK、PI-3K、NO-cGMP、Rho、NF-κB通路。当AT2受体被PD123319阻断时,64个基因表达差异在2倍以上,48个下调,16个上调;11条途径基础活化,其中7个基因的改变在30倍以上：Cyp19a1（37倍）、I1lr2（42倍）、Cflar（53倍）、Bcl21（31倍）、Pik3cg（278倍）、Cdknla（90倍）、Agt（162倍）。在AT1受体阻断的基础上再阻断AT2受体,46个基因表达差异在2倍以上,36个下调,10个上调;11条信号途径全部活化。其结果与单独阻断AT2受体信号途径基本一致。RT-PCR选取IL-1β和TNF-α进行验证,结果与芯片各组间的变化趋势基本相符。结果表明,在成年大鼠心肌成纤维细胞,AT2受体阻断明显不同于AT1受体阻断,在信号转导通路相关基因表达谱上,两者有显著差异。     

Conversion of angiotensin I to angiotensin II by cathepsin A isoenzymes of porcine kidney

We have reported the existence of a carboxypeptidase in a human renal extract that converts Angiotensin I (AI) to Angiotensin II (AII) in two steps with des-leu-AI (dl-AI) being formed as an intermediate. Since this carboxypeptidase had properties similar to cathepsin A, the ability of cathepsin A to metabolize AI was studied. Cathepsin A was purified from hog kidney with enzyme activity being monitored using both benzyloxycarbonyl-glutamyl-tyrosine (ZGT) and AI as substrates. The procedure separated the expected large and small molecular weight forms of cathepsin A as well as two additional isoenzymes. All of the isoenzymes had carboxypeptidase activity with ZGT, AI, and dl-AI. No detectable cleavage of AII was observed. Cathepsin A,S (small) activity with ZGT or AI as substrate was inhibited to a similar extent by diisopropylfluorophosphate, mersalyl acid, and a decapeptide renin inhibitor. It is concluded that the renal angiotensin carboxypeptidase activity is catalyzed by cathepsin A. By its ability to convert AI to AII, cathepsin A may be a component of the intrarenal renin-angiotensin system.     

Salt crystal deposition as a reversible mechanism to enhance photoprotection in black mangrove

Mangrove forests are ecosystems made up of several woody plants living in saline coastal sedimentary habitats. In order to deal with the high salinity of the substrate, mangrove trees possess a number of different mechanisms to exclude, sequestrate or excrete the excess of salt. The black mangrove (Avicennia germinans L.), one of the dominant species in Central America, is characterized by high levels of salt excretion through epidermal glands. In this study, our aim was to examine whether, apart from its obvious role in salt tolerance, the formation of salt crystals on the upper leaf surface of black mangrove might represent an unusual and dynamic photoprotection mechanism. For this purpose, the reflection of light and a number of physiological parameters were studied during the dry and rainy seasons in black mangroves growing in the Juan Venado Island Nature Reserve (Nicaragua). Excreted salt increased the reflectance of the leaf surface mainly in the blue and red regions of the spectrum. By removing salt crust from the leaf surface, we demonstrated that during the most stressful periods (dry season at noon), this feature allowed leaves to maintain a higher photochemical efficiency and a lower leaf temperature as compared to uncovered leaves. Furthermore, this mechanism is fully reversible when conditions become more favorable, as salt crystals dissolve, forming drops. Thus, while being a detoxification mechanism developed mainly to avoid osmotic imbalance in the tissues, the excretion of salt through the leaves in black mangroves is an example of “exaptation”, as it has positive collateral effects on the photosynthetic performance of the plant, protecting A. germinans from overheating and photodamage during the harsher periods.     

Degradation of fodrin by m-calpain in fibroblasts adhering to fibrillar collagen I gel

When skin fibroblasts were cultured on fibrillar collagen I gel, we observed rapid degradation of talin, fodrin and ezrin, which are well-known calpain substrates. The protease m-calpain was activated only in cells adhering to fibrillar collagen, whereas micro-calpain was activated in cells adhering to monomeric or fibrillar collagen at the same level. The calpain inhibitor Z-Leu-Leu-aldehyde inhibited degradation of fodrin, but not talin. Degradation of fodrin, alpha-actinin and ezrin was prevented by over-expression of dominant negative m-calpain. However, over-expression of calpastatin, an endogenous calpain inhibitor, had no effect the degradation of these three proteins. These results suggest that m-calpain is responsible for degradation of their membrane proteins via adhesion to fibrillar collagen I gel.     

Smad3 mediates the TGF-beta-induced contraction of type I collagen gels by mouse embryo fibroblasts

TGF-beta signals through TGF-beta receptors and Smad proteins. TGF-beta also augments fibroblast-mediated collagen gel contraction, an in vitro model of connective tissue remodeling. To investigate the importance of Smad2 or Smad3 in this augmentation process, embryo-derived fibroblasts from mice lacking expression of Smad2 or Smad3 genes were cast into native type I collagen gels. Fibroblast-populated gels were then released into 0.2% FCS-DMEM alone or with recombinant human TGF-beta1, beta2, beta3, or recombinant rat PDGF-BB. Gel contraction was determined using an image analyzer. All three isoforms of TGF-beta significantly augmented contraction of collagen gels mediated by fibroblasts with genotypes of Smad2 knockout (S2KO), Smad2 wildtype (S2WT), and Smad3 wildtype (S3WT), but not Smad3 knockout (S3KO) mice. PDGF-BB augmented collagen gel contraction by all fibroblast types. These results suggest that expression of Smad3 but not Smad2 may be critical in TGF-beta augmentation of fibroblast-mediated collagen gel contraction. Thus, the Smad3 gene could be a target for blocking contraction of fibrotic tissue induced by TGF-beta.