Fibroblast Activation Protein Cleaves and Inactivates Fibroblast Growth Factor 21

FGF21 is a stress-induced hormone with potent anti-obesity, insulin-sensitizing, and hepatoprotective properties. Although proteolytic cleavage of recombinant human FGF21 in preclinical species has been observed previously, the regulation of endogenously produced FGF21 is not well understood. Here we identify fibroblast activation protein (FAP) as the enzyme that cleaves and inactivates human FGF21. A selective chemical inhibitor, immunodepletion, or genetic deletion of Fap stabilized recombinant human FGF21 in serum. In addition, administration of a selective FAP inhibitor acutely increased circulating intact FGF21 levels in cynomolgus monkeys. On the basis of our findings, we propose selective FAP inhibition as a potential therapeutic approach to increase endogenous FGF21 activity for the treatment of obesity, type 2 diabetes, non-alcoholic steatohepatitis, and related metabolic disorders.     

Predictors of diagnosis and survival in idiopathic pulmonary fibrosis and connective tissue disease-related usual interstitial pneumonia

Background

Although usual interstitial pneumonia (UIP) appears to portend better survival when associated with connective tissue disease (CTD-UIP), little is known about the presenting clinical, radiologic, and pathologic features that differentiate pathologically confirmed UIP with CTD from idiopathic pulmonary fibrosis (IPF). In patients with atypical radiologic and clinical features, what specific findings predict underlying IPF vs. CTD-UIP diagnosis and their respective long term survival?

Methods

A large retrospective cohort analysis was done of consecutive patients seen from 1995 through 2010 with biopsy confirmed UIP completed or reviewed at our institution. CTD-UIP was defined by independent rheumatology consultation with exclusion of all other secondary causes of lung fibrosis. Primary clinical data was collected and compared for IPF and CTD-UIP along with logistic regression performed for predictors of disease likelihood and Cox proportional hazards analysis for predictors of survival.

Results

Six hundred and twenty five patients were included in the study of which 89 had diagnosed CTD-UIP representing 7 disease entities. Survival was better among those with CTD-UIP except in UIP associated with rheumatoid arthritis, which had similar presenting features and survival to IPF. Predictors of underlying CTD included female gender, younger age, positive autoimmune serology, and inconsistent presenting radiologic findings. Only age and forced vital capacity corrected for a priori covariates were predictive of survival in CTD-UIP.

Conclusions

UIP pathology occurs frequently among patients with atypically presenting clinical and radiologic features, and may represent IPF or CTD-UIP with improved prognosis if underlying CTD is diagnosed. Presenting radiologic and pathologic features alone are not predictive of underlying secondary cause or survival between the two groups.     

Severe Extracellular Matrix Abnormalities and Chondrodysplasia in Mice Lacking Collagen Prolyl 4-Hydroxylase Isoenzyme II in Combination with a Reduced Amount of Isoenzyme I

Collagen prolyl 4-hydroxylases (C-P4H-I, C-P4H-II, and C-P4H-III) catalyze formation of 4-hydroxyproline residues required to form triple-helical collagen molecules. Vertebrate C-P4Hs are α₂β₂ tetramers differing in their catalytic α subunits. C-P4H-I is the major isoenzyme in most cells, and inactivation of its catalytic subunit (P4ha1^−/−) leads to embryonic lethality in mouse, whereas P4ha1^+/− mice have no abnormalities. To study the role of C-P4H-II, which predominates in chondrocytes, we generated P4ha2^−/− mice. Surprisingly, they had no apparent phenotypic abnormalities. To assess possible functional complementarity, we established P4ha1^+/−;P4ha2^−/− mice. They were smaller than their littermates, had moderate chondrodysplasia, and developed kyphosis. A transient inner cell death phenotype was detected in their developing growth plates. The columnar arrangement of proliferative chondrocytes was impaired, the amount of 4-hydroxyproline and the T_m of collagen II were reduced, and the extracellular matrix was softer in the growth plates of newborn P4ha1^+/−;P4ha2^−/− mice. No signs of uncompensated ER stress were detected in the mutant growth plate chondrocytes. Some of these defects were also found in P4ha2^−/− mice, although in a much milder form. Our data show that C-P4H-I can to a large extent compensate for the lack of C-P4H-II in proper endochondral bone development, but their combined partial and complete inactivation, respectively, leads to biomechanically impaired extracellular matrix, moderate chondrodysplasia, and kyphosis. Our mouse data suggest that inactivating mutations in human P4HA2 are not likely to lead to skeletal disorders, and a simultaneous decrease in P4HA1 function would most probably be required to generate such a disease phenotype.     

Identification of an Endogenously Generated Cryptic Collagen Epitope (XL313) That May Selectively Regulate Angiogenesis by an Integrin Yes-associated Protein (YAP) Mechano-transduction Pathway

Extracellular matrix (ECM) remodeling regulates angiogenesis. However, the precise mechanisms by which structural changes in ECM proteins contribute to angiogenesis are not fully understood. Integrins are molecules with the ability to detect compositional and structural changes within the ECM and integrate this information into a network of signaling circuits that coordinate context-dependent cell behavior. The role of integrin αvβ3 in angiogenesis is complex, as evidence exists for both positive and negative functions. The precise downstream signaling events initiated by αvβ3 may depend on the molecular characteristics of its ligands. Here, we identified an RGD-containing cryptic collagen epitope that is generated in vivo. Surprisingly, rather than inhibiting αvβ3 signaling, this collagen epitope promoted αvβ3 activation and stimulated angiogenesis and inflammation. An antibody directed to this RGDKGE epitope but not other RGD collagen epitopes inhibited angiogenesis and inflammation in vivo. The selective ability of this RGD epitope to promote angiogenesis and inflammation depends in part on its flanking KGE motif. Interestingly, a subset of macrophages may represent a physiologically relevant source of this collagen epitope. Here, we define an endothelial cell mechano-signaling pathway in which a cryptic collagen epitope activates αvβ3 leading to an Src and p38 MAPK-dependent cascade that leads to nuclear accumulation of Yes-associated protein (YAP) and stimulation of endothelial cell growth. Collectively, our findings not only provide evidence for a novel mechano-signaling pathway, but also define a possible therapeutic strategy to control αvβ3 signaling by targeting a pro-angiogenic and inflammatory ligand of αvβ3 rather than the receptor itself.     

Molecular cloning and expression analysis of chymotrypsin-like serine protease from the redclaw crayfish (Cherax quadricarinatus): A possible role in the junior and adult innate immune systems

A novel chymotrypsin-like serine protease (CLSP) was isolated from the hepatopancreas of the redclaw crayfish Cherax quadricarinatus (Cq-chy). The full-length cDNA of Cq-chy contains 951 nucleotides encodes a peptide of 270 amino acids. The mature peptide comprising 223 amino acids contains the conserved catalytic triad (H, D, and S). Similarity analysis showed that Cq-chy shares high identity with chymotrypsins from the fiddler crab; Uca pugilator. Cq-chy mRNA expression in C. quadricarinatus was shown to be: (a) tissue-related with the highest expression in the hepatotpancreas and widely distributed, (b) highly responsive in the hepatopancreas to White Spot Syndrome Virus (WSSV) challenge, and (c) differently regulated in immature and adult crayfish. In this study we successfully isolated Cq-chy. Our observations indicate that Cq-chy is differently involved in the immature and adult innate immune reactions, thus suggesting a role for CLSPs in the invertebrate innate immune system.     

Involvement of gelatinases (MMP-2 and MMP-9) in the development of airway inflammation and pulmonary fibrosis

Pulmonary fibrosis has an aggressive course and is usually fatal an average of 3 to 6 years after the onset of symptoms. Pulmonary fibrosis is associated with deposition of extracellular matrix (ECM) components in the lung interstitium. Matrix metalloproteinases (MMPs) are a major group of proteinases known to regulate the ECM remodeling and so they are hypothesized to be important in the process of lung fibrosis. These led to the concept that modulation of airway remodeling including excessive proteolytic damage of the tissue may be of interest for future treatment. The excessive airway remodeling as a result of an imbalance in the equilibrium of the normal processes of synthesis and degradation of extracellular matrix components could argue in favor of antiprotease treatments. Moreover, these observations emphasize that effective therapies for these disorders must be given early in the natural history of the disease, prior to the development of expensive lung destruction and fibrosis. This revised version was published online in August 2006 with corrections to the Cover Date.     

Procollagen C-endopeptidase Enhancer Protein 2 (PCPE2) Reduces Atherosclerosis in Mice by Enhancing Scavenger Receptor Class B1 (SR-BI)-mediated High-density Lipoprotein (HDL)-Cholesteryl Ester Uptake

Studies in human populations have shown a significant correlation between procollagen C-endopeptidase enhancer protein 2 (PCPE2) single nucleotide polymorphisms and plasma HDL cholesterol concentrations. PCPE2, a 52-kDa glycoprotein located in the extracellular matrix, enhances the cleavage of C-terminal procollagen by bone morphogenetic protein 1 (BMP1). Our studies here focused on investigating the basis for the elevated concentration of enlarged plasma HDL in PCPE2-deficient mice to determine whether they protected against diet-induced atherosclerosis. PCPE2-deficient mice were crossed with LDL receptor-deficient mice to obtain LDLr^−/−, PCPE2^−/− mice, which had elevated HDL levels compared with LDLr^−/− mice with similar LDL concentrations. We found that LDLr^−/−, PCPE2^−/− mice had significantly more neutral lipid and CD68+ infiltration in the aortic root than LDLr^−/− mice. Surprisingly, in light of their elevated HDL levels, the extent of aortic lipid deposition in LDLr^−/−, PCPE2^−/− mice was similar to that reported for LDLr^−/−, apoA-I^−/− mice, which lack any apoA-I/HDL. Furthermore, LDLr^−/−, PCPE2^−/− mice had reduced HDL apoA-I fractional clearance and macrophage to fecal reverse cholesterol transport rates compared with LDLr^−/− mice, despite a 2-fold increase in liver SR-BI expression. PCPE2 was shown to enhance SR-BI function by increasing the rate of HDL-associated cholesteryl ester uptake, possibly by optimizing SR-BI localization and/or conformation. We conclude that PCPE2 is atheroprotective and an important component of the reverse cholesterol transport HDL system.     

Reduction-oxidation (Redox) and vascular tissue level of homocyst(e)ine in human coronary atherosclerotic lesions and role in extracellular matrix remodeling and vascular tone

Hyperhomocyst(e)inemia in patients with coronary and peripheral arterial occlusion has been demonstrated by others. Redox-state of homocyst(e)ine causes dysfunction of endothelial cells and promote growth of vascular smooth muscle cells. The role of tissue, protein bound and unbound, oxidative mixed disulfides in the development of fibrous plaque in atherosclerotic lesion is not known. Redox-state around the fibroblasts and vascular smooth muscle cells modulates the expression of extracellular matrix (ECM) components (Tyagi et al. 1996, J Cell Biochem, 61: 139-151). To determine the role of tissue homocystine in fibrotic atherosclerotic plaque development, coronary arteries were isolated from ischemic explanted hearts (n = 10). Apparently normal vascular tissue was obtained from idiopathic cardiomyopathic explanted hearts (n = 10). Tissue extract were prepared from atherosclerotic lesions and from normal arteries devoid of adventitia. Interaction of homocystine with Ellman's reagent (5, 5-dithio-bis-2-nitro benzoic acid) catalyzed by limiting amount of reducing agent (catalyst) generated change in optical density (OD) at 412 nm in dose dependent fashion. We have generated a standard curve between change at 412 nm and amount of homocystine. The change in OD at 412 nm with increasing amount (0-25 g) of homocystine demonstrated linearity. The protein-bound oxidized disulfides were precipitated by trichloroacetic acid (TCA) and free-oxidative disulfides in the supernatant were collected. The pathophysiological amount of protein-bound disulfide in atherosclerotic tissue (1.0 ± 0.2g/mg total protein) was 10 times that in normal tissue (0.1 ±0.01 g/mg, p < 0.001). The amount of free oxidative disulfide in atherosclerotic tissue (1.5 ± 0.3g/mg) was 15 times that in normal tissue (0.12 ± 0.02 g/mg, p < 0.001). To determine the role of homocystine in ECM expression, ECM collagenase activity in the presence and absence of homocystine was measured by zymography. The effect of homocysteine on collagenase activity was biphasic, increased at < [0.01 mM] and inhibited at > [0.1 mM]. To determine whether homocystine regulates vascular tone, isometric measurements were carried out using normal coronary rings. Results suggested that homocystine induced endothelial-modulated vasoconstriction in coronary vessels. Tissue oxidative disulfides and the homocystine may contribute to the development of fibrotic atherosclerotic lesions and vascular dysfunction.     

Nitrotyrosinylation,remodeling and endothelial‐myocyte uncoupling in iNOS,cystathionine beta synthase (CBS) knockouts and iNOS/CBS double knockout mice

Increased levels of homocysteine (Hcy), recognized as hyperhomocysteinemia (HHcy), were associated with cardiovascular diseases. There was controversy regarding the detrimental versus cardio protective role of inducible nitric oxide synthase (iNOS) in ischemic heart disease. The aim of this study was to test the hypothesis that the Hcy generated nitrotyrosine by inducing the endothelial nitric oxide synthase, causing endothelial‐myocyte (E‐M) coupling. To differentiate the role of iNOS versus constitutive nitric oxide synthase (eNOS and nNOS) in Hcy‐mediated nitrotyrosine generation and matrix remodeling in cardiac dysfunction, left ventricular (LV) tissue was analyzed from cystathionine beta synthase (CBS) heterozygote knockout, iNOS homozygote knockout, CBS?/+/iNOS?/? double knockout, and wild‐type (WT) mice. The levels of nitrotyrosine, MMP‐2 and ‐9 (zymographic analysis), and fibrosis (by trichrome stain) were measured. The endothelial‐myocyte function was determined in cardiac rings. In CBS?/+ mice, homocysteine was elevated and in iNOS?/? mice, nitric oxide was significantly reduced. The nitrotyrosine and matrix metalloproteinase‐9 (MMP‐9) levels were elevated in double knockout and CBS?/+ as compared to WT mice. Although MMP‐2 levels were similar in CBS?/+, iNOS?/?, and CBS?/+/iNOS?/?, the levels were three‐ to fourfold higher than WT. The levels of collagen were similar in CBS?/+ and iNOS?/?, but they were threefold higher than WT. Interesting, the levels of collagen increased sixfold in double knockouts, compared to WT, suggesting synergism between high Hcy and lack of iNOS. Left ventricular hypertrophy was exaggerated in the iNOS?/? and double knockout, and mildly increased in the CBS?/+, compared to WT mice. The endothelial‐dependent relaxation was attenuated to the same extent in the CBS?/+ and iNOS?/?, compared to WT, but it was robustly blunted in double knockouts. The results concluded that homocysteine generated nitrotyrosine in the vicinity of endothelium, caused MMP activation and endothelium‐myocyte uncoupling. The generation of nitrotyrosine was independent of iNOS. J. Cell. Biochem. 106: 119–126, 2009. © 2008 Wiley‐Liss, Inc.     

The Tyrosine Kinase c-Abl Promotes Homeodomain-interacting Protein Kinase 2 (HIPK2) Accumulation and Activation in Response to DNA Damage

The non-receptor tyrosine kinase c-Abl is activated in response to DNA damage and induces p73-dependent apoptosis. Here, we investigated c-Abl regulation of the homeodomain-interacting protein kinase 2 (HIPK2), an important regulator of p53-dependent apoptosis. c-Abl phosphorylated HIPK2 at several sites, and phosphorylation by c-Abl protected HIPK2 from degradation mediated by the ubiquitin E3 ligase Siah-1. c-Abl and HIPK2 synergized in activating p53 on apoptotic promoters in a reporter assay, and c-Abl was required for endogenous HIPK2 accumulation and phosphorylation of p53 at Ser⁴⁶ in response to DNA damage by γ- and UV radiation. Accumulation of HIPK2 in nuclear speckles and association with promyelocytic leukemia protein (PML) in response to DNA damage were also dependent on c-Abl activity. At high cell density, the Hippo pathway inhibits DNA damage-induced c-Abl activation. Under this condition, DNA damage-induced HIPK2 accumulation, phosphorylation of p53 at Ser⁴⁶, and apoptosis were attenuated. These data demonstrate a new mechanism for the induction of DNA damage-induced apoptosis by c-Abl and illustrate network interactions between serine/threonine and tyrosine kinases that dictate cell fate.     

Single and Compound Knock-outs of MicroRNA (miRNA)-155 and Its Angiogenic Gene Target CCN1 in Mice Alter Vascular and Neovascular Growth in the Retina via Resident Microglia

The response of the retina to ischemic insult typically leads to aberrant retinal neovascularization, a major cause of blindness. The epigenetic regulation of angiogenic gene expression by miRNAs provides new prospects for their therapeutic utility in retinal neovascularization. Here, we focus on miR-155, a microRNA functionally important in inflammation, which is of paramount importance in the pathogenesis of retinal neovascularization. Whereas constitutive miR-155-deficiency in mice results in mild vascular defects, forced expression of miR-155 causes endothelial hyperplasia and increases microglia count and activation. The mouse model of oxygen-induced retinopathy, which recapitulates ischemia-induced aberrant neovessel growth, is characterized by increased expression of miR-155 and localized areas of microglia activation. Interestingly, miR-155 deficiency in mice reduces microglial activation, curtails abnormal vessel growth, and allows for rapid normalization of the retinal vasculature following ischemic insult. miR-155 binds to the 3′-UTR and represses the expression of the CCN1 gene, which encodes an extracellular matrix-associated integrin-binding protein that both promotes physiological angiogenesis and harnesses growth factor-induced abnormal angiogenic responses. Single CCN1 deficiency or double CCN1 and miR-155 knock-out in mice causes retinal vascular malformations typical of faulty maturation, mimicking the vascular alterations of miR-155 gain of function. During development, the miR-155/CCN1 regulatory axis balances the proangiogenic and proinflammatory activities of microglia to allow for their function as guideposts for sprout fusion and anastomosis. Under ischemic conditions, dysregulated miR-155 and CCN1 expression increases the inflammatory load and microglial activation, prompting aberrant angiogenic responses. Thus, miR-155 functions in tandem with CCN1 to modulate inflammation-induced vascular homeostasis and repair.     

Fibronectin enhances viability and alters cytoskeletal functions (with effects on the phosphatidylinositol 3-kinase pathway) in small cell lung cancer

Small cell lung cancer (SCLC) is a rapidly progressive disease with ultimate poor outcome. SCLC has been shown to interact closely with the stromal and extracellular matrix (ECM) components of the diseased host. ECM consists of type I/IV collagen, laminin, vitronectin, and fibronectin (FN) among others. Herein, we investigated the behavior of a SCLC cell line (NCI-H446) on FN-coated surface. Over a course of 72 h, FN (10 micro g/ml) caused both increased survival and proliferation of NCI-H446 cells. Survival under serum-starved conditions increased 1.44-fold and proliferation in the presence of fetal calf serum increased by 1.30-fold. The phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 reduced both survival and proliferation of NCI-H446 cells (0.48- and 0.27-fold, respectively), even on FN-coated surface. We next determined the effects of FN on cytoskeletal function such as cell motility/morphology and adhesion. Over a course of 24 h, FN reduced aggregation of NCI-H446 cells and induced flattened cellular morphology with neurite-like projections after 1 h, however, in the presence of LY294002, the cells rounded up. Adhesion of NCI-H446 cells also increased with FN (4.47-fold) which was abrogated with LY294002 treatment. This correlated with phosphorylation of the cytoskeletal protein p125FAK, on Tyr397, Tyr861 and Ser843 residues with FN. Even in the presence of LY294002, these serine/tyrosine residues were still phosphorylated on FN-coated surface. In contrast, the focal adhesion protein paxillin was not phosphorylated at Tyr31 with FN. In summary, FN stimulation of SCLC cells leads to enhancement of viability and changes in cytoskeletal function that are partially mediated through the PI3-K pathway.     

Conserved phosphorylation of serines in the Ser-X-Glu/Ser(P) sequences of the vitamin K-dependent matrix Gla protein from shark, lamb, rat, cow, and human.

The present studies demonstrate that matrix Gla protein (MGP), a 10-kDa vitamin K-dependent protein, is phosphorylated at 3 serine residues near its N-terminus. Phosphoserine was identified at residues 3, 6, and 9 of bovine, human, rat, and lamb MGP by N-terminal protein sequencing. All 3 modified serines are in tandemly repeated Ser-X-Glu sequences. Two of the serines phosphorylated in shark MGP, residues 2 and 5, also have glutamate residues in the n + 2 position in tandemly repeated Ser-X-Glu sequences, whereas the third, shark residue 3, would acquire an acidic phosphoserine in the n + 2 position upon phosphorylation of serine 5. The recognition motif found for MGP phosphorylation, Ser-X-Glu/Ser(P), has been seen previously in milk caseins, salivary proteins, and a number of regulatory peptides. A review of the literature has revealed an intriguing dichotomy in the extent of serine phosphorylation among secreted proteins that are phosphorylated at Ser-X-Glu/Ser(P) sequences. Those phosphoproteins secreted into milk or saliva are fully phosphorylated at each target serine, whereas phosphoproteins secreted into the extracellular environment of cells are partially phosphorylated at target serine residues, as we show here for MGP and others have shown for regulatory peptides and the insulin-like growth factor binding protein 1. We propose that the extent of serine phosphorylation regulates the activity of proteins secreted into the extracellular environment of cells, and that partial phosphorylation can therefore be explained by the need to ensure that the phosphoprotein be poised to gain or lose activity with regulated changes in phosphorylation status.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipopolysaccharide Induction of MARCKS-Related Protein and Cytokine Secretion are Differentially Impaired in Microglia from LPS-nonresponsive (C3H/HeJ) Mice

Many events involved in activation of microglia and leukocytes by lipopolysaccharide (LPS) are mediated by protein kinase C (PKC), and we have recently demonstrated that a major PKC substrate, MARCKS-related protein (MRP), is selectively induced by LPS in murine microglia. In microglia from LPS-nonresponsive (C3H/HeJ) mice, induction of MRP and secretion of CSF-1 required much higher LPS concentrations (100 ng/ml) than in normal (C3H/OuJ) microglia (10 ng/ml). By contrast, TNF production was not significantly increased in C3H/HeJ microglia even at 1 g LPS/ml. Microglia expressed PKC isoforms , , , and (but not and ); PKC isoform levels were similar in both normal and C3H/HeJ microglia and no significant change in response to LPS was noted. Our results indicate that LPS alters PKC substrate (rather than kinase) expression, and that the Lps^d mutation in C3H/HeJ mice differentially affects regulation of several gene products implicated in microglial function.     

Transient receptor potential (TRP) channels as molecular targets in lung toxicology and associated diseases

The lungs as the gateways of our body to the external environment are essential for gas exchange. They are also exposed to toxicants from two sides, the airways and the vasculature. Apart from naturally produced toxic agents, millions of human made chemicals were produced since the beginning of the industrial revolution whose toxicity still needs to be determined. While the knowledge about toxic substances is increasing only slowly, a paradigm shift regarding the proposed mechanisms of toxicity at the plasma membrane emerged. According to their broad-range chemical reactivity, the mechanism of lung injury evoked by these agents has long been described as rather unspecific. Consequently, therapeutic options are still restricted to symptomatic treatment. The identification of molecular down-stream effectors in cells was a major step forward in the mechanistic understanding of the action of toxic chemicals and will pave the way for more causal and specific toxicity testing as well as therapeutic options. In this context, the involvement of Transient Receptor Potential (TRP) channels as chemosensors involved in the detection and effectors of toxicant action is an attractive concept intensively discussed in the scientific community. In this review we will summarize recent evidence for an involvement of TRP channels (TRPA1, TRPC4, TRPC6, TRPV1, TRPV4, TRPM2 and TRPM8) expressed in the lung in pathways of toxin sensing and as mediators of lung inflammation and associated diseases like asthma, COPD, lung fibrosis and edema formation. Specific modulators of these channels may offer new therapeutic options in the future and will endorse strategies for a causal, specifically tailored treatment based on the mechanistic understanding of molecular events induced by lung-toxic agents.