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Basement membranes are thin sheets of extracellular matrix with many diverse roles in the body. Those in normal tissue are also highly insoluble and resist attempts to extract and characterize their components. A mouse tumor, the EHS tumor, has provided large amounts of basement membrane material, which has facilitated the structural and functional characterization of its components. An extract of the tumor, known as Matrigel, contains components which reconstitute into a solid gel at 37°. This solid basement membrane matrix has been used in both cell culture and in vivo. Matrigel has been utilized in some 12,000-plus publications for a variety of studies with embryonic, normal, and stem or malignant cells. Evidence presented in this Commentary suggests that Matrigel isolated from tumors grown in diverse hosts could exert unique effects that could be helpful in analyzing the causes of various pathologies and for screening possible therapeutic agents.

Objective To provide guidance about management of psoriatic disease during the COVID-19 pandemic. Study Design A task force (TF) of 18 physician voting members with expertise in dermatology, rheumatology, epidemiology, infectious diseases, and critical care was convened. The TF was supplemented by non-voting members which included fellows and National Psoriasis Foundation (NPF) staff. Clinical questions relevant to the psoriatic disease community were informed by questions received by the NPF. A Delphi process was conducted. Results The TF approved 22 guidance statements. The average of the votes was within the category of agreement for all statements. All guidance statements proposed were recommended, 9 with high consensus, 13 with moderate consensus. Limitations The evidence behind many guidance statements is limited in quality. Conclusion These statements provide guidance for the management of patients with psoriatic disease on topics ranging from how the disease and its treatments impact COVID-19 risk and outcome, how medical care can be optimized during the pandemic, what patients should do to lower their risk of getting infected with SARS-CoV-2 and what they should do if they develop COVID-19. The guidance is intended to be a living document that will be updated by the TF as data emerge., Capsule Summary The NPF COVID-19 Task Force produced 22 guidance statements to promote optimal management of psoriatic disease during the pandemic. Shared decision making is recommended as is adherence to evidence-based recommendations when available. The guidance statements will be updated when necessary in accordance with rapidly evolving science of COVID-19.

Objective To update guidance regarding the management of psoriatic disease during the COVID-19 pandemic. Study Design The task force (TF) includes 18 physician voting members with expertise in dermatology, rheumatology, epidemiology, infectious diseases, and critical care. The TF was supplemented by non-voting members, which included fellows and National Psoriasis Foundation (NPF) staff. Clinical questions relevant to the psoriatic disease community were informed by inquiries received by the NPF. A Delphi process was conducted. Results The TF updated evidence for the original 22 statements and added 5 new recommendations. The average of the votes was within the category of agreement for all statements, 13 with high consensus, 14 with moderate consensus. Limitations The evidence behind many guidance statements is variable in quality and/or quantity. Conclusion These statements provide guidance for the management of patients with psoriatic disease on topics ranging from how the disease and its treatments impact COVID-19 risk, how medical care can be optimized during the pandemic, what patients should do to lower their risk of getting infected with SARS-CoV-2 (including novel vaccination), and what they should do if they develop COVID-19. The guidance is a living document that is continuously updated by the TF as data emerge., Capsule Summary:• The NPF COVID-19 TF produced 27 guidance statements to promote optimal management of psoriatic disease during the pandemic. • Shared decision making, adherence to evidence-based treatment, and the urgent use of novel COVID-19 vaccination are recommended. The guidance statements will be updated in accordance with the rapidly evolving science of COVID-19.

Background/Aims Systemic sclerosis (SSc) is a rare and progressive connective tissue disease that is more common in women in the third to fifth decades of life and is rare in children. Calcinosis cutis, the deposition of calcium deposits within the subcutaneous tissue, remains a challenging non-lethal complication of SSc. The development of calcinosis cutis is a poorly understood area and there are no functional mouse models or laboratory models for calcinosis in SSc. In this study we present clinical database analysis plus two potential in vitro models for examining calcinosis in the setting of SSc. Methods Clinical modelling through database analysis of n = 79 SSc patients with and without calcinosis was attempted. In tissue culture studies, the first model system utilised adipose-derived mesencyhmal stem cells (MSCs) stimulated with interstitial fluid from healthy controls or SSc patients (both n = 4). In a second model, macrophages from patients with SSc (n = 4 lines) were used to stimulate MSCs in co-culture. We examined the impact that this has on the deposition of calcium hydroxyapatite production after prolonged culture in osteogenic media, measured using a scale (scored 0-4) to describe the degree of alizarin red uptake. We also investigated the role of an inhibitor of TGFβ signalling, SB432542 (10µM), and RP832c,(10µM), a peptide therapy which targets and repolarises activated SSc macrophages via CD206, in reducing calcium hydroxyapatite production. Results Clinical database analysis demonstrated that 35% of patients had evidence of calcinosis, but the only significant factor between the calcinosis and non-calcinosis group was the presence of digital ulcerations (p = 0.0008). The tissue fluid based model of calcinosis demonstrated that on day 21 the blister fluid obtained from SSc patients induced a significant increase in calcium deposition as compared to the culture media alone (3.9 vs 2.5, p = 0.00071), whereas SB431542, reduced calcium production by SSc fluid treated cells (3.9 vs 3.0, p = 0.2147). In the cell based model, macrophages derived from SSc patients induced the production of mineralised deposits. Both the TGFβ inhibitor SB432542 and the RP832c peptide reduced calcinosis in this model (SB431542 0.5 vs 1.3 p = 0.0547, RP832c 0.2 vs 1.3 p = 0.0147). Conclusion Our understanding of the underlying pathogenesis of calcinosis within SSc remains poor leading to therapeutic challenges for clinicians. From this analysis, it appears that presence of digital ulceration is associated clinically with calcinosis, possibly implicating aberrant tissue repair or ischaemia. Both tissue culture models presented here may recreate certain aspects of calcinosis in patients with SSc and may provide a basis for further evaluation of therapeutic inhibitors. At present there are no therapies that have been consistently found to be effective but our data support a possible role for macrophage - mesenchymal stem cell interactions and for TGFβ. Disclosure B.G.T. Coumbe: None. S.N. Ahmad: None. G. Thomas: None. L. Borukhson: None. B. Ahmed Abdi: None. H. Lopez: None. C. Garvin: None. J. Jaynes: None. C. Yates: None. G. Martin: None. C. Denton: None. D. Abraham: None. R.J. Stratton: None.

Activated M2 polarized macrophages are drivers of pulmonary fibrosis in several clinical scenarios such as Acute Respiratory Disease Syndrome (ARDS) and Idiopathic Pulmonary Fibrosis (IPF), through the production of inflammatory and fibrosis-inducing cytokines. In this study, we investigated the effect of targeting the CD206 receptor with a novel fragment of a Host Defense Peptide (HDP), RP-832c to decrease cytokines that cause fibrosis. RP-832c selectively binds to CD206 on M2 polarized bone marrow derived macrophages (BMDM) in vitro, resulting in a time-dependent decrease in CD206 expression, and a transient increase in M1 marker TNFα, which resolves over a 24hr period. To elucidate the antifibrotic effect of RP-832c, we used a murine model of bleomycin (BLM) -induced early-stage pulmonary fibrosis. RP-832c significantly reduced bleomycin-induced fibrosis in a dosage dependent manner, as well as decreased CD206, TGF-β1 and α-SMA expression in mouse lungs. Interestingly we did not observe any changes in the resident alveolar macrophage marker CD170 expression. Similarly, in an established model of lung fibrosis, RP-832c significantly decreased fibrosis in the lung, as well as significantly decreased inflammatory cytokines TNFα, IL-6, IL-10, INF-γ, CXCL1/2, and fibrosis markers TGF-β1 and MMP-13. In comparison with FDA approved drugs, Nintedanib and Pirfenidone, RP-832c exhibited a similar reduction in fibrosis compared to Pirfenidone, and to a greater extent than Nintedanib, with no apparent toxicities observed on body weight or blood chemistry. In summary, RP-832c is a potential agent to mitigate the overactivity of M2 macrophages in pathogenesis several pulmonary fibrotic diseases, including SARS-CoV-2 induced lung fibrosis.

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Introduction Clinical diversity in systemic sclerosis (SSc) reflects multifaceted pathogenesis and the effect of key growth factors or cytokines operating within a disease-specific microenvironment. Dermal interstitial fluid sampling offers the potential to examine local mechanisms and identify proteins expressed within lesional tissue. We used multiplex cytokine analysis to profile the inflammatory and immune activity in the lesions of SSc patients. Methods Dermal interstitial fluid sample from the involved forearm skin, and synchronous plasma samples were collected from SSc patients (n = 26, diffuse cutaneous SSc (DcSSc) n = 20, limited cutaneous SSc (LcSSc) n = 6), and healthy controls (HC) (n = 10) and profiled by Luminex® array for inflammatory cytokines, chemokines, and growth factors. Results Luminex® profiling of the dermal blister fluid showed increased inflammatory cytokines (median interleukin ( IL)-6 in SSc 39.78 pg/ml, HC 5.51 pg/ml, p = 0.01, median IL-15 in SSc 6.27 pg/ml, HC 4.38 pg/ml, p = 0.03), chemokines (monocyte chemotactic protein (MCP)-3 9.81 pg/ml in SSc, 7.18 pg/ml HC, p = 0.04), and profibrotic growth factors (platelet derived growth factor (PDGF)-AA 10.38 pg/ml versus 6.94 pg/ml in HC, p = 0.03). In general dermal fluid and plasma cytokine levels did not correlate, consistent with predominantly local production of these factors within the dermal lesions, rather than leakage from the serum. In hierarchical clustering and network analysis IL-6 emerged as a key central mediator. Conclusions Our data confirm that an immuno-inflammatory environment and aberrant vascular repair are intimately linked to fibroblast activation in lesional skin in SSc. This non-invasive method could be used to profile disease activity in the clinic, and identifies key inflammatory or pro-fibrotic proteins that might be targeted therapeutically. Distinct subgroups of SSc may be defined that show innate or adaptive immune cytokine signatures.

The basement membrane extracellular matrix contacts epithelial, endothelial, fat and smooth muscle cells. Because this extracellular matrix is so thin, it had been hard to study its composition, structure, and function. An extract of a tumor was found to contain all of the components present in basement and to be very biologically active. This extract, termed Matrigel, Cultrex, or EHS matrix, promotes cell differentiation, and is used to measure the invasive activity of tumor cells. In vivo, it is used for measuring angiogenic inhibitors and stimulators, to improve graft survival, repair damaged tissues, and increase tumor growth.

A simplification of the laminin nomenclature is presented. Laminins are multidomain heterotrimers composed of alpha, beta and gamma chains. Previously, laminin trimers were numbered with Arabic numerals in the order discovered, that is laminins-1 to -5. We introduce a new identification system for a trimer using three Arabic numerals, based on the alpha, beta and gamma chain numbers. For example, the laminin with the chain composition alpha5beta1gamma1 is termed laminin-511, and not laminin-10. The current practice is also to mix two overlapping domain and module nomenclatures. Instead of the older Roman numeral nomenclature and mixed nomenclature, all modules are now called domains. Some domains are renamed or renumbered. Laminin epidermal growth factor-like (LE) domains are renumbered starting at the N-termini, to be consistent with general protein nomenclature. Domain IVb of alpha chains is named laminin 4a (L4a), domain IVa of alpha chains is named L4b, domain IV of gamma chains is named L4, and domain IV of beta chains is named laminin four (LF). The two coiled-coil domains I and II are now considered one laminin coiled-coil domain (LCC). The interruption in the coiled-coil of beta chains is named laminin beta-knob (Lbeta) domain. The chain origin of a domain is specified by the chain nomenclature, such as alpha1L4a. The abbreviation LM is suggested for laminin. Otherwise, the nomenclature remains unaltered.

Idiopathic pulmonary fibrosis (IPF) is an insidious inflammatory fibroproliferative disease whose cause and course before diagnosis are unknown, and for which existing treatments are of limited benefit. The National Heart, Lung, and Blood Institute convened a working group to develop specific recommendations for future IPF research. Inflammatory and immune processes are involved in IPF pathogenesis, and current therapeutic strategies are aimed at suppressing the inflammation. Recent data suggest that the molecular processes underlying the fibrogenesis may provide new opportunities for therapeutic intervention. Specific areas of future research recommended by the working group include studies to elucidate the etiology of IPF, to develop novel diagnostic techniques and molecular diagnostics, to establish a program for identification of molecular targets for IPF treatment and identification and generation of agonists or antagonists that inhibit fibrogenesis, to foster investigations that couple the use of new technologies (e.g., laser capture microdissection, microarrays, and mass spectroscopic analysis of proteins) with data from the human genome project, to establish a national consortium of Clinical Centers of Excellence to conduct coordinated clinical and laboratory studies of well-characterized patients and patient-derived materials, and to stimulate research to develop animal models of persistent and progressive pulmonary fibrosis for evaluation of new intervention approaches.

Objective: To evaluate biomechanical properties of skin in patients with systemic sclerosis (SSc) using the BTC-2000 suction device. Methods: Twenty five patients with limited cutaneous SSc (lcSSc), 20 patients with diffuse disease (dcSSc), and 25 age matched healthy controls were evaluated. Viscoelastic deformation (VED, mm), elastic deformation (ED, mm), ultimate deformation (UD, mm), and pressure-deformation ratio (PDR, mm Hg/mm) were measured on the dorsal surface of the forearm, shoulder, and above the trapezius muscle on the back. Results: Indices of skin extensibility (VED, ED, UD) were reduced and resistance to stress (PDR) increased in patients with dcSSc compared with healthy controls, or patients with lcSSc, at all three sites (p

Keloids are locally exuberant dermal scars characterized by excessive fibroblast proliferation and matrix accumulation. Although treatment strategies include surgical removal and intralesional steroid injections, an effective regimen is yet to be established due to a high rate of recurrence. The regressing center and growing margin of the keloid have different collagen architecture and also differ in the rate of proliferation. To investigate whether proliferation is responsive to collagen topography, keloid, scar, and dermal fibroblasts were cultured on nanopatterned scaffolds varying in collagen fibril diameter and alignment-small and large diameter, aligned and random fibrils, and compared to cells grown on flat collagen-coated substrates, respectively. Cell morphology, proliferation, and expression of six genes related to proliferation (cyclin D1), phenotype (α-smooth muscle actin), and matrix synthesis (collagens I and III, and matrix metalloproteinase-1 and -2) were measured to evaluate cell response. Fibril alignment was shown to reduce proliferation and matrix synthesis in all three types of fibroblasts. Further, keloid cells were found to be most responsive to nanotopography.

A series of oriented electrospun nylon fibers were prepared with nominal fiber diameters of either 300 nm or 1 m. In addition, a series of similar mats were prepared that contained a hyperbranched polymer additive to act as a surface modifier for the fibers. The mats were used as reinforcements for epoxy composites. The composite coupons were interrogated using techniques such as dynamic mechanical analysis and tensile testing. While the nanofibers did not dramatically stiffen the resulting composites, they provided insight as to the impact of the additives on the interaction of the fibers with the composite matrix. The inclusion of the additive appeared to improve interfacial interactions of the fibers with the matrix, as reduced void content and uniform fracture interfaces were observed in the modified composites. While the low production rate of suitable fiber mats for this study limited the scope and accessibility of test specimens, the results from the examination of fiber-matrix interfaces argues for further investigation.

Patients with scleroderma receiving Iloprost as a treatment for severe Raynaud's phenomenon report a reduction in skin tightness, suggesting that this drug inhibits skin fibrosis. Connective tissue growth factor (CTGF), a recently described profibrotic cytokine, acts downstream and in concert with TGF-beta to stimulate the fibrotic process and is involved in the fibrosis seen in scleroderma. Here we show that Iloprost, acting by elevation of cAMP, blocks the induction of CTGF and the increase in collagen synthesis in fibroblasts exposed to TGF-beta. The potency of Iloprost with respect to suppression of CTGF far exceeds that of other prostanoid receptor agonists, suggesting that its effect is mediated by the prostacyclin receptor IP. By sampling dermal interstitial fluid using a suction blister device, we show that CTGF levels are greatly elevated in the dermis of scleroderma patients compared with healthy controls and that Iloprost infusion causes a marked decrease in dermal CTGF levels. These studies suggest that Iloprost could be reducing the level of a key profibrotic cytokine in scleroderma patients and that endogenous production of eicosanoids may limit the fibrotic response to TGF-beta.

We have used representational difference analysis (RDA) to identify up-regulated genes in skin fibroblasts from fibrotic lesions obtained from patients with systemic sclerosis (scleroderma). RDA of cDNA libraries derived from fibroblasts from involved and uninvolved skin detected several differentially expressed genes. One such gene consistently up-regulated in scleroderma cells coded for human connective tissue growth factor (CTGF). Other studies described here show that the CTGF protein is readily detected in cultures of systemic sclerosis fibroblasts but was not detected in comparable normal cells. High levels of CTGF are also evident in biological fluids from patients with systemic sclerosis. TGFbeta stimulates CTGF production in both normal and systemic sclerosis fibroblasts with the latter found to be higher producers. Moreover, an analysis of constitutive and TGFbeta-induced CTGF gene activation showed altered and elevated transcriptional responses in systemic sclerosis cells compared with controls. CTGF stimulated a two- to threefold increase in proalpha1(I) collagen and fibronectin synthesis by both dermal and lung fibroblasts in culture and promoted significant matrix remodeling of fibroblast-populated three-dimensional collagen lattices. A direct relation between the overexpression of CTGF and elevated collagen synthesis was suggested by the observation that transfection of a CMV-CTGF cDNA construct and protein expression in fibroblasts increased the transcription of a Col 1alpha2 promoter-reporter construct to levels seen in systemic sclerosis fibroblasts. Using Col 1alpha2 promoter deletion constructs the CTGF responsive element was localized to the first 379 bp upstream of the transcriptional start site. These data indicate that there is an overexpression of CTGF in the systemic sclerosis cells, probably due to increased gene transcription, and suggest that the dysregulation of CTGF production is an important factor in fibroblast activation and the excessive deposition of collagen in systemic sclerosis.

Multiple lines of evidence suggest that macrophages, in particular M2-polarized tumor associated macrophages (TAMs), promote tumor aggressiveness and chemoresistance. Thus, limiting the pro-tumorigenic activity of TAMs may hold great therapeutic promise. To assess this potential, we synthesized artificial peptides (10-12mers) with a striapathic arrangement of hydrophobic and hydrophilic amino acids, designed to specifically target TAMs within the tumor microenvironment. These peptides, including drug candidate RP-182, are basic in design and bind to human albumin prior to binding to their eventual target, which enhances peptide half-life to around 2 hours. In vitro experiments demonstrated that exposure to 50uM peptide significantly reduced M2 macrophage viability and increased apoptosis after 48 hours of exposure, but had no effect on M1 macrophages. Interestingly, we did not observe a significant effect of RP-182 in vitro on various cancer cell lines, including MDA-MB-231, C42B or cells isolated from KPC960 mice. To determine whether RP-182 had in vivo efficacy we used multiple animal models, beginning with inflammatory indications where macrophage activity is also implicated, and progressing to solid organ cancer models: (1) In bleomycin animal models of lung and skin fibrosis, mice developed very high levels of inflammatory markers including IL6, which remained at control levels in mice treated with 10 mg/kg RP-182 qD subcu. At increased pulmonary bleomycin levels, all control animals died while 65% of treated animals survived. (2) In xenograft models of triple negative breast cancer (MDA-MB-231) mice treated with peptide (10 mg/kg qD subcu) demonstrated 37% reduction in MD-MB-231 tumor growth; gemcitabine @ 40 mg/kg q4D demonstrated 70% reduction; however, peptide plus gemcitabine completely blocked tumor growth. (3) In a xenograft model of castration resistant prostate cancer (CRPC) (C42B) mice treated with 2.5 mg/kg Docetaxel q7D demonstrated 45% reduction in tumor growth relative to control; mice treated with 10 mg/kg RP-182 qD demonstrated 52% reduction in tumor growth; but Docetaxel plus RP-182 reduced tumor growth 65%. (4) In the KPC960 model of pancreatic cancer, RP-182 treatment caused a 30% reduction in tumor growth and reduced the expression of a number of inflammatory genes in the tumors. (5) Finally, in the KRAS/p53 transgenic model of pancreatic cancer, treatment with peptide for one week reduced tumor growth, and was associated with 5.8 fold reduction in PDL1 and a 5.5 fold reduction in PDL2 expression in tumors. Toxicity testing on animals given much higher doses of RP-182 showed no changes in histology, blood counts, body or tissue weight. These results suggest that these novel designed peptides suppress macrophages in the tumors, enhancing the tumor response to chemotherapy and downregulating certain checkpoint proteins. This abstract is also presented as Poster B23. Citation Format: Henry Lopez, George R. Martin, Jesse M. Jaynes, Clayton Yates. Synthetic peptides suppress M2 macrophages and synergize with chemotherapy in prostate and breast cancer models. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr PR03.

This report describes the construction of an optical test system to measure the electro-optic coefficients of poled polymers directly. The poling process in polymers is the spontaneous orientation of the dissolved dye molecule, the chromophore, in the presence of an applied electric field. This spontaneous orientation confers upon the poled polymer a variety of useful optical effects, the linear electro-optic effect being chief among them. The optical test system measures the electro-optic coefficient directly by measuring the relative retardance of one polarization, the p-polarization, relative to the other polarization present, the s-polarization. This method has advantages over previous methods that have relied on measuring the very weak second-harmonic signal emitted from poled polymers. This report describes the measurement of the electro-optic coefficient of a number of electro-optic chromophores synthesized in the Weapons and Materials Research Directorate (WMRD) and intended for use in electro-optic modulation devices.

Aging is characterized by numerous physical, physiological, biochemical, and molecular changes. The rates at which aging processes occur are highly variable among individuals and are thought to be governed by both environmental and genetic factors. Lifestyle factors such as exercise, dietary, and smoking habits have been demonstrated to alter many of the changes usually associated with human aging. However, at present caloric restriction is the only experimental paradigm that has consistently been demonstrated in animal models to extend not only physiological vigor but also life span. The positive effects of exercise on physiological fitness and the reduction in the risks of certain diseases have been well documented. However, its effects on life span are not as clear. This article explores some of the basic mechanisms thought to be involved causally in the processes of aging, and outlines current and potential interventive strategies to retard or ameliorate the rates of decline in physiological function with advancing age.

Primary human dermal fibroblasts isolated from the medial aspect of the proximal forearm of young and old donors were compared for the expression of interstitial collagenase, 72 kDa type IV collagenase, the tissue inhibitor of metalloproteinase type 1, and pro-α2 (I) collagen mRNA at basal levels and after stimulation with the tumor promotor 12- O -tetradecanoyl-phorbol-13-acetate. Higher basal and induced steady-state mRNA levels of interstitial collagenase were found in the cells from older donors. Ratios of basal and induced steady-state mRNA levels of interstitial collagenase to pro-α2 (I) collagen, and interstitial collagenase to the tissue inhibitor of metalloproteinases type 1 were also higher in the cells from older donors. Seventy-two kiloDalton type IV collagenase and pro-α2 (I) collagen mRNA showed similar levels of expression in the cells from young and old donors and were not altered by treatment with 12- O -tetradecanoyl-phorbol-13-acetate. Transient transfection assays with the interstitial collagenase promoter linked to a reporter gene showed increased activity of the reporter in cell strains with high interstitial collagenase mRNA levels. Mobility shift assays demonstrated increased binding activity to the specific 12- O -tetradecanoyl-phorbol-13-acetate response element in nuclear extracts from the cell strains with higher induced collagenase mRNA levels and higher reporter gene activity. These findings are consistent with the observed phenotype of interstitial collagenase and its specific tissue inhibitor in the senescent fibroblast aging model.

In this study, we investigated how matrix nanotopography affects corneal fibroblast phenotype and matrix synthesis. To this end, corneal fibroblasts isolated from bovine corneas were grown on collagen nanofiber scaffolds of different diameters and alignment--30 nm aligned fibrils (30A), 300 nm or larger aligned fibrils (300A), and 30 nm nonaligned fibrils (30NA) in comparison with collagen coated flat glass substrates (FC). Cell morphology was visualized using confocal microscopy. Quantitative PCR was used to measure expression levels of six target genes: the corneal crystallin-transketolase (TKT), the myofibroblast marker-α-smooth muscle actin (SMA), and four matrix proteins-collagen 1 (COL1), collagen 3 (COL3), fibronectin (FN), and biglycan. It was found that SMA expression was down-regulated and TKT expression was increased on all three collagen nanofiber substrates, compared with the FC control substrates. However, COL3 and biglycan expression was also significantly increased on 300A, compared with the FC substrates. Thus matrix nanotopography down-regulates the fibrotic phenotype, promotes formation of the quiescent keratocyte phenotype, and influences matrix synthesis. These results have significant implications for the engineering of corneal replacements and for promoting regenerative healing of the cornea after disease and/or injury.

Looking in the right place with the right assay was the key to discovering collagenase and the first matrix metalloprotease (MMP) in the pregenomic era. Gross and Lapiere (1962) cultured living pieces of metamorphosing tadpole tail in collagen gels and observed lyses of the gel around the fragment, even when the tissue was separated from the collagen by a filter. This suggested that a diffusible protease was produced by living tadpole tissue. Similar studies were conducted with tumor tissue, bone, and human fibroblasts, and so on, also showing collagenase activity. EDTA inhibited cleavage suggesting an MMP and serum also inhibited enzyme activity. Remarkably, collagen was cleaved at a single site, leaving intact 4 and 1 4 fragments of the molecule. Gross (2004) and Lapiere (2005) have recently revisited their discovery and subsequent events. It is interesting that the tadpole, as well as mice and rats, lack the collagenase 1 (MMP1) gene and utilize other members of the family. The tadpole collagenase 4 gene contains thyroxine response elements, which may coordinate metamorphosis and the resorption of collagen Fujimoto et al. (2006). Lazarus et al. (1968), in attempting to explain how collagen was degraded during inflammation, extracted a collagenase stored in human granulocytes (MMP8), which differed in certain properties from the tadpole enzyme. With the discovery of genetically distinct collagens, Liotta et al. (1979) found that malignant tumor cells secreted an enzyme (MMP2), which degraded basement membrane (type IV) collagen, but not collagens I, II, III or V. This suggested that the production of this enzyme by malignant tumor cells allowed them to cross basement membrane barriers and disseminate through the body. The requirement for a different collagenase to degrade basement membranes would also allow retention of tissue shape during remodeling and growth. This enzyme has also been referred to as gelatinase A based on its activity in an assay developed by Harris and Krane (1972), which used labeled, denatured collagen to detect proteases in rheumatoid synovial fluid. Indeed, MMP2 cleaves a number of proteins other than collagen IV and a variety of MMPs also degrade collagen IV, dangerous to generalize. The selectivity of certain MMPs as collagenases is likely to relate to their affinity for helical collagens and their ability to unwind a less rigid helical site on the molecule and cleave there, thus attacking their Achilles’ heel. An important development in the genomic era was the isolation of a cDNA clone coding for an MMP having a putative transmembrane domain (membrane type MMP1, mtMMP1/MMP14) (Sato et al., 1994). Transfection of this MMP cDNA into cells produced an MMP located on the cell surface, demonstrated that it activated proMMP2 and that cells expressing MMP14 had an enhanced ability to migrate through basement membrane barriers. Several such MMPs have now been described and they appear to have many diverse functions, including activating proMMPs secreted by other cells and degrading collagen and other matrix components by direct cell contact. Much remains unsettled. Why so many MMPs with overlapping activities? Given the great diversity of collagens, do certain MMPs account for the removal of certain collagens? MMP13/collagenase 3 has been implicated in cartilage turnover as well as in rheumatoid arthritis and osteoarthritis (Inada et al., 2004). A variety of MMPS are implicated in tumor growth, spread and during angiogenesis. Cleavage of a portion of laminin 5 was shown to convert it from an attachment factor to a stimulator of migration (Giannelli et al., 1997), with obvious relevance to wound healing and cancer spread. The enzyme mammalian tolloid was shown to be the enzyme in the skin that performs this function in human skin (Veitch et al., 2003). The MMP system resembles a cascade. Growth requires tissue turnover and the removal of a variety of connective tissue components, which vary from tissue to tissue. Fragments of matrix proteins often have activities of their own, which regulate the process. It is far more complicated than expected and has not yet, but certainly will, yield therapeutic solutions.

We transfected mouse 10T1/2 fibroblasts with the H-ras oncogene and isolated lines expressing H-ras. One of the lines exhibited a highly malignant phenotype with the ability to produce large tumors and to colonize the lung after tail vein injection. In addition, the cells of this line showed increased collagenase IV production, directed migration and invasiveness, properties associated with the ability of tumor cells to metastasize. Since cyclic adenosine 3',5'-monophosphate (cAMP) is known to down-regulate ras expression, we exposed the malignant cells (Cl-1) to either N6, 2',0-dibutyryl cAMP (DB-cAMP) or 8-bromo cAMP (8-Br-cAMP), either with or without a phosphodiesterase inhibitor. We found that these treatments reduced the expression of ras, chemotaxis, invasiveness, and lung colonization of the ras-transformed cells. We therefore postulate that the malignancy of some cells may be regulated by alterations in the intracellular cAMP levels by suppressing ras expression and/or by reducing other activities required for the dissemination of tumor cells.

Lack of estrogen receptor (ER) and presence of vimentin (VIM) associate with poor prognosis in human breast cancer. We have explored the relationships between ER, VIM, and invasiveness in human breast cancer cell lines. In the matrigel outgrowth assay, ER+/VIM- (MCF-7, T47D, ZR-75-1), and ER-/VIM- (MDA-MB-468, SK-Br-3) cell lines were uninvasive, while ER-/VIM+ (BT549, MDA-MB-231, MDA-MB-435, MDA-MB-436, Hs578T) lines formed invasive, penetrating colonies. Similarly, ER-/VIM+ cell lines were significantly more invasive than either the ER+/VIM- or ER-/VIM- cell lines in the Boyden chamber chemoinvasion assay. Invasive activity in nude mice was only seen with ER-/VIM+ cell lines MDA-MB-231, MDA-MB-435 and MDA-MB-436. Hs578T cells (ER-/VIM+) showed hematogenous dissemination to the lungs in one of five mice, but lacked local invasion. The ER-/VIM+ MCF-7ADR subline was significantly more active than the MCF-7 cells in vitro, but resembled the wild-type MCF-7 parent in in vivo activity. Data from these cell lines suggest that human breast cancer progression results first in the loss of ER, and subsequently in VIM acquisition, the latter being associated with increased metastatic potential through enhanced invasiveness. The MCF-7ADR data provide evidence that this transition can occur in human breast cancer cells. Vimentin expression may provide useful insights into mechanisms of invasion and/or breast cancer cell progression.

Previously we found that the reconstituted basement membrane matrix Matrigel, when premixed with human small-cell lung carcinoma cells and injected subcutaneously into athymic mice, permitted tumor growth, whereas cells injected in the absence of Matrigel did not form tumors. In the present study, we examined additional cell types and determined some of the underlying mechanisms involved in the promotion of tumor formation by Matrigel. The tumor cell lines that we studied included transformed mouse Englebreth-Holm-Swarm tumor cells (T-EHS), human submandibular carcinoma A253 cells, mouse melanoma B16F10 cells, human epidermoid carcinoma KB cells, and human primary renal cell carcinoma cells. When coinjected subcutaneously with Matrigel, these cell lines formed rapidly proliferating tumors. Primary biopsy specimens of human colon carcinoma, when dispersed and coinjected with Matrigel, also formed tumors. Only A253, KB, and B16F10 cells formed small tumors in the absence of Martrigel, but a fivefold to tenfold increase in tumor size was observed in the presence of Matrigel. These data demonstrate a useful method for improving the growth of human tumors in athymic mice.

The laminin A chain has been sequenced by cDNA cloning and was found to contain an RGD sequence. Synthetic peptides containing the RGD sequence and flanking amino acids were active in mediating cell adhesion, spreading, migration, and neurite outgrowth. Furthermore, endothelial cell attachment to a laminin substrate was inhibited by an RGD-containing synthetic peptide. Antisera against the integrin (fibronectin) receptor, and monoclonal antibody to the integrin, VLA-6, inhibited cell interaction with laminin, as well as with peptides containing an RGD sequence. These results suggest that the RGD containing site of laminin is active and interacts with the integrin family of receptors in certain cells.

Epidermal growth factor (EGF) expression in the congenital polycystic mouse kidney. The mechanisms responsible for renal cyst formation in congenital polycystic kidney disease remain unknown, although abnormalities of cellular metabolism, basement membrane components, and growth factors have been suggested. In the present study, we examined a potential role for epidermal growth factor (EGF) in cyst formation in a mouse model. We measured growth factor activity and concentration in renal cyst fluid, urine, and serum obtained from mice with congenital polycystic kidney disease (cpk). In affected mice, both growth factor activity of urine and the urinary EGF concentration were much lower than unaffected littermate controls even when corrected for creatinine concentration. Although the growth factor activity was much lower in affected mice, there were significant differences in the regional distribution of EGF in animals with cysts. Both growth factor activity and EGF concentration were greater in cyst fluid when compared to urine. Growth factor activity in cysts was completely inhibited by anti-EGF antibody using BALB/MK epithelial keratinocytes as targets. The expression of EGF mRNA in kidneys from affected mice was markedly decreased when compared to littermate controls. Theses results suggest that decreased EGF production and local differences in EGF concentration may contribute to cyst formation.

Several constituents of basement membranes, including type IV collagen, laminin, heparan sulphate proteoglycan and nidogen, form a defined supramolecular complex that is an obligatory intermediate in the formation of this matrix. We have named this defined supramolecular complex the 'basement membrane matrisome'. Matrisome structures composed of other collagens, proteoglycans and glycoproteins may participate in the formation of other extracellular matrices. Cells show specific interactions with components of the extracellular matrix. We discuss studies that indicate that melanoma cells can express receptors for both laminin and fibronectin. However, these receptors are expressed in a reciprocal fashion, depending on the exposure of the cell to these proteins. Binding of either fibronectin or laminin to the cells elicits a distinct phenotype. This represents a mechanism in which cellular activity can be regulated by extracellular matrix factors during development and in repair.

Small cell lung cancer (SCLC) is a fatal malignancy due to its propensity to metastasize widely and to reoccur after chemotherapy in a drug-resistant form. While most SCLC cell lines are anchorage independent for growth, laminin induced the attachment of five of six SCLC cell lines tested (NCI-N417, NCI-H345, NCI-H146, NCI-H187, NCI-H510, and NCI-H209). NCI-N417 SCLC cells adopted a flattened morphology on laminin, and a classic SCLC cell line (NCI-H345) demonstrated a neuron-like appearance while the other SCLC cell lines except NCI-H187 cells, attached but did not spread. Adhesion to laminin was associated with increased resistance to several cytotoxic drugs. Matrigel, an extract of basement membrane proteins, greatly accelerated tumor growth when coinjected with SCLC cells in athymic mice. A synthetic peptide from the B1 chain of laminin, cyclic-YIGSR (Tyr-Ile-Gly-Ser-Arg), inhibited laminin-induced SCLC cell adhesion and migration in vitro and reduced the size of the tumors they formed when coinjected with matrigel and YIGSR. These results suggest that the interaction of SCLC cells with laminin and possibly with other basement membrane proteins can enhance their tumorigenicity and drug resistance.

Altered basement membrane protein biosynthesis by primary cultures of cpk/cpk mouse kidney. Previously, kidneys from three-week-old cpk/cpk C57/B16 mice were found to contain elevated mRNA levels for the basement membrane components collagen IV and laminin [1]. Here primary cultures of kidney epithelial cells derived from cpk/cpk C57/B16 mice were established and the production of these proteins in culture was studied. Primary cultures of cpk/cpk mouse kidney epithelial cells were observed to have a more polygonal, flattened morphology than cells from unaffected littermate kidneys. The rate of collagen IV and laminin biosynthesis was determined by means of [ 35 S] labelling studies followed by immunoprecipitation. Collagen IV and laminin biosynthesis are elevated by approximately twofold or more in primary cultures derived from 20-day-old cpk/cpk mice, as compared with parallel primary cultures derived from their unaffected littermates. Similarly, laminin Bl chain mRNA is elevated in primary cultures derived from 20-day-old cpk/cpk mice. In primary cultures derived from younger (day 11) mice, similar differences in the rates of both collagen and laminin biosynthesis were not observed between the two culture types. These observations are consistent with the previously reported age-dependent differences observed in laminin and in collagen IV gene expression in both cpk/cpk and wild-type mouse kidneys, and suggest that the regulation of overproduction of these proteins is due to an alteration in the kidney cells and not due to systemic factors.

Tumor cells attach, degrade, and migrate through basement membranes as they metastasize. Laminin, a major glycoprotein of basement membranes, promotes the metastatic activity of tumor cells by stimulating the attachment and migration of the cells and their secretion of collagenase IV. We have identified a synthetic peptide of 19 amino acids (Cys-Ser-Arg-Ala-Arg-Lys-Gln-Ala-Ala-Ser-Ile-Lys-Val-Ala-Val-Ser-Ala-Asp -Arg) from the sequence of the A chain of laminin that increases experimental metastases of the lungs by murine melanoma cells. The peptide is active when injected either intravenously or intraperitoneally. The peptide increased collagenase IV activity, a key enzyme in the breakdown of basement membranes, to the same extent as laminin. This peptide represents an active site on laminin for promotion of the metastatic phenotype and generates a probe for studying the regulation of malignant activities.

The invasive activities of some malignant cells appear to be activated by contact with laminin. This protein occurs solely in basement membranes and the binding of malignant cells to the surface of this extracellular matrix initiates the invasion process. Passage of the cells across basement membrane requires degradative activity and laminin induces the production of collagenase IV which lyses the collagen IV network. The motility of the cells is enhanced by chemo-attractants and by matrix molecules. Peptides that inhibit the binding of tumour cells to laminin, inhibitors of collagenase IV, and inhibitors of specific pathways of arachidonic acid metabolism prevent invasion as well as the metastasis of malignant cells and could be employed to stop the spread of cancer.

The authors have adopted a new nomenclature for the laminins. They are numbered with arabic numerals in the order discovered. The previous A, B1 and B2 chains, and their isoforms, are alpha, beta and gamma, respectively, followed by an arabic numeral to identify the isoform. For example, the first laminin identified from the Engelbreth-Holm-Swarm tumor is laminin-1 with the chain composition alpha 1 beta 1 gamma 1. The genes for these chains are LAMA1, LAMB1 and LAMC1, respectively.

prescribed HPN, which is life-saving in patients with severe bowel involvement. Our study shows that HPN offers a safe means of nutritional support for patients with severe SSc-related GI involvement, but that SSc-related mortality remains high. The catheter-related sepsis rate of patients with SSc was low. In addition, the majority of patients with SSc relied on others for their catheter care. Disclosure statement: The authors have declared no conflicts of interest.

Chronologic and biologic aging begin at conception, although senescent changes, defined here as functional declines, are usually apparent only after sexual maturation.1 With time, physiologic decrements become increasingly more common and increasingly compromise health and survival. Particularly notable is a reduced capacity with age to adjust to a variety of everyday stresses, probably due to defects in basic homeostatic mechanisms.2 The rates of aging and the life span for any species are governed by two general features: a genetic component that imparts a species-specific capability to carry out basic biologic processes necessary for life and reproduction and environmental, and lifestyle components that are superimposed on the intrinsic genetic design and can influence the overall rates of aging and susceptibility to disease processes.3

Metastasis is a complex process, almost a cascade, involving multiple steps and activities. However, an important factor is that malignant cells are able to penetrate through the multiple basement membrane barriers surrounding tissues, blood vessels, nerves and muscle that would otherwise block their dissemination. Penetration of malignant tumor cells through basement membrane is an active process requiring proteolysis. We report here that inhibitors of both the cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism convert mouse melanoma and human fibrosarcoma cells to a non invasive state by reducing the production of MMP-2, an enzyme required for the degradation of basement membranes. Specific metabolites of each pathway, i.e. PGF2 alpha and 5-HPETE, are able to transcend the block and restore collagenase production, invasiveness in vitro and metastatic activity in vivo. These studies indicate a key role for arachidonic acid metabolites in metastasis and suggest novel therapeutic approaches for inhibiting the spread of cancer.

Background Cancer incidence increases with age, but the growth and spread of tumors is often slow and prolonged in the elderly. Transplantable murine tumors grow and spread less readily in older mice and can be used as models to study the effect of host age. Neovascularization is crucial for the growth of solid tumors, and alterations in the host vascular response may underlie the changes in tumor growth occurring with age. Purpose We have used transplantable tumor cells and tumors, and tumor extracts, to better understand differences in the biology of tumor growth and vascularization as a function of host age. Methods Englebreth-Holm-Swarm (EHS) carcinoma and B16-F10 melanoma cells were injected into C57BL mice of different ages. Tumor growth, histology, and cellular DNA synthesis were compared. Vascularization was determined using basic fibroblast growth factor and an in vivo angiogenesis assay. EHS tumor extracts were assayed for biologic activity in vitro using human endothelial cells and in vivo using mouse EHS-BAM carcinoma and human TSU-Pr1 prostate carcinoma cells. Results EHS tumors formed larger tumors in young than in old C57BL mice. Rapid tumor growth resumed upon transfer of tumor tissue from old animals into young animals. The rate of DNA synthesis of tumor tissue from old animals in organ culture was lower than in tissue from young animals. Histologically, tumors grown in old animals exhibited a threefold higher ratio of extracellular matrix to tumor cells than those grown in young animals. Tumors from adult animals exhibited numerous small blood vessels; those from old animals contained fewer, much larger vessels. Similar results were observed in young mice fed a reduced-calorie diet. Young animals elicited a greater and more rapid angiogenic response than old animals. Extracts of tumors grown in old animals failed to support endothelial cell differentiation in culture. Tumor cells injected together with such old extracts showed reduced tumor growth in nude mice. Conclusions The rate of growth and morphology of the EHS tumor were altered with age, partly due to a reduced capacity to vascularize the tumors because of a lack of angiogenic factors or the presence of host inhibitors. Implications Alterations in host factors detected in this tumor model may underlie a variety of age-dependent changes that could influence tumor growth and the repair and regeneration of normal tissue. Reducing the vascularization of tumors represents a potential target to reduce their growth and progression.

Three stable carcinoma cell lines, designated RM22-F5, RM17-5R, and RM1-4, were established from spontaneously occurring mammary carcinomas in old, outbred, female Wistar rats. The RM22-F5 and RM17-5R cells were keratin-positive and formed epithelial monolayers, whereas RM1-4 cells exhibited a spindle-like morphology and intense vimentin staining. When injected into nude mice, RM22-F5, RM17-5R and RM1-4 cells formed well-differentiated, poorly differentiated and undifferentiated carcinomas, respectively. The relative growth rates of the tumor cells in vitro were RM1-4 > RM22-F5 > RM17-5R. The growth of RM22-F5, but not of RM17-5R and RM1-4 cells, was significantly stimulated by insulin, epidermal growth factor, dexamethasone, 17 beta-estradiol and progesterone in vitro. Ovariectomy reduced the growth of RM22-F5 cells in vivo and these cells (but not RM1-4 or RM17-5R) were estrogen-receptor (ER)-positive. None of the lines were positive for the progesterone receptor (PR). Spontaneous lung and lymph-node metastases were observed in nude mice injected with RM22-F5 or RM17-5R cells, respectively. In contrast, RM1-4 cells were non-metastatic but invasive. Karyotype analysis revealed that RM22-F5 cells were hyperdiploid, RM17-5R were hypotetraploid, and RM1-4 were diploid with a sizeable insertion in chromosome 1. A point mutation in codon 12 (G to A transition) and loss of the normal allele of the H-ras-1 gene was detected in the DNA from RM22-F5 cells. No p53 mutations were apparent in any of the cell lines. The results indicate that RM22-F5 cells are hormone-dependent with an ER+/PR- phenotype, while the RM17-5R and RM1-4 lines are hormone-independent and ER-/PR-. These cell lines exhibit the spectrum of biological properties and genetic alterations observed in human breast cancers and may, therefore, be novel and useful models for understanding sporadic breast cancer in post-menopausal women.