Bioactive Fragments Research Articles

Matricryptins derived from collagens and proteoglycans

Controlled proteolysis of extracellular matrix components releases bioactive fragments or unmasks cryptic sites that play key roles in controlling various physio-pathological processes including angiogenesis, tissue remodeling, wound healing, inflammation, tumor growth, and metastasis. We review here the structure and mechanisms of release of i) the proteolytic fragments (matricryptins) cleaved from collagens, proteoglycans and glycosaminoglycans, and ii) the matricryptic sites existing in these molecules. The cell surface receptors and the signaling pathways they trigger to exert their biological activities is discussed with the major physio-pathological processes they control. Their involvement in autoimmune and inherited diseases is reported. Most matricryptins issued from collagens, proteoglycans and glycosaminoglycans exhibit anti-angiogenic and anti-tumor properties and their use as potential drugs and as potential disease markers is discussed. Perspectives for identifying the common structural features, if any, of the matricryptins and their use in combination with chemotherapy and radiotherapy in the treatment of cancer are presented.

Bioactive peptides derived from vascular endothelial cell extracellular matrices promote microvascular morphogenesis and wound healing in vitro

Studies in our laboratory indicate that collagenase from Clostridium histolyticum promotes endothelial cell and keratinocyte responses to injury in vitro and wound healing in vivo. We postulate that matrix degradation by Clostridial collagenase creates bioactive fragments that can stimulate cellular responses to injury and angiogenesis. To test this hypothesis, we performed limited digestion of defined capillary-endothelial-derived extracellular matrices using purified human or bacterial collagenases. Immunoprecipitation with antibodies recognizing collagens I, II, III, IV, and V, followed by mass spectrometry reveals the presence of unique fragments in bacterial, but not human-enzyme-digested matrix. Results show that there are several bioactive peptides liberated from Clostridial collagenase-treated matrices, which facilitate endothelial responses to injury, and accelerate microvascular remodeling in vitro. Fragments of collagen IV, fibrillin-1, tenascin X, and a novel peptide created by combining specific amino acids contained within fibrillin 1 and tenascin X each have profound proangiogenic properties. The peptides used at 10-100 nM increase rates of microvascular endothelial cell proliferation by up to 47% and in vitro angiogenesis by 200% when compared with serum-stimulated controls. Current studies are aimed at revealing the molecular mechanisms regulating peptide-induced wound healing while extending these in vitro observations using animal modeling.

Author Response: Bevacizumab Suppression of Establishment of Micrometastases in Experimental Ocular Melanoma

Bevacizumab is a recombinant humanized monoclonal IgG1 antibody that binds to and inhibits the biological activity of human vascular endothelial growth factor (VEGF) in in vitro and in vivo assay systems. It contains human framework regions and the complementarity-determining regions of a murine antibody that binds to VEGF. When comparing the sequence of human VEGF protein with that of mouse VEGF protein, we found that 86% of the protein sequence in mouse VEGF164 is similar to that of human VEGF165.1 We believe that it is possible for bevacizumab to block human and mouse VEGF. A blocking antibody is defined as an antibody that does not have a reaction when combined with an antigen, but prevents other antibodies from combining with that antigen.2,3 We believe that bevacizumab fits this definition and explains why the neutralized VEGF is not detected by ELISA. The human or mouse VEGF immunoassay (Quantikine; R&D Systems, Minneapolis, MN) that we used in our experiment can detect human VEGF165 and VEGF121 or mouse VEGF164 and VEGF120, but does not detect VEGF189 and VEGF206. VEGF121 and VEGF165 are diffusible proteins that are secreted into the medium. VEGF189 and VEGF206 have high affinity for heparin and are mostly bound to heparin-containing proteoglycans in the extracellular matrix. Bevacizumab binds to and neutralizes all isoforms of VEGF-A and bioactive proteolytic fragments. Bock et al.4 performed three different experimental approaches using Western blot analysis, ELISA, and Biacore (surface plasmon resonance; GE Health Care, Piscataway, NJ) analysis to assess whether bevacizumab binds to murine VEGF-A. In the Western blot analyses, recombinant murine VEGF-A164, a 24-kDa protein consisting of 164 amino acid residues, and the 19.1-kDa homologous human VEGF165, which consists of 165 amino acid residues, were used. In ELISA and the Biacore analyses, the recombinant human and the homologous murine VEGF-A were used as well. Western blot analysis showed that bevacizumab bound murine and human VEGF-A. To confirm these results, Bock et al. performed two different experimental approaches for analysis of the binding of bevacizumab to murine VEGF-A. The first assay, an ELISA, analyzed the interaction of soluble bevacizumab with VEGF-A immobilized on a microtiter plate surface. In the second assay, the surface plasmon resonance technology (Biacore) was used for the analysis of interaction of soluble VEGF-A with bevacizumab immobilized on the sensor chip surface. Both assays allow a qualitative characterization of the interaction between bevacizumab and human or murine VEGF-A. In the ELISA binding assay, bevacizumab bound to both human and murine VEGF-A; however, a 1000-fold higher bevacizumab concentration was necessary to reach a similar level of binding to murine VEGF-A compared with that needed for binding to the human protein. There was no significant binding of isotype control IgG1k to murine VEGF-A at this concentration. During the Biacore surface plasmon resonance experiments, different concentrations of human and murine VEGF-A were injected, to analyze their binding to immobilized bevacizumab. A more than 10-fold higher concentration of murine VEGF-A than of human VEGF-A was necessary to reach similar resonance units. ELISA is more sensitive than Western blot analysis. It can detect a protein of at picogram concentrations; Western blot can only detect a protein at nanogram concentrations. Studies have also indicated that bevacizumab inhibits experimental choroidal and corneal neovascularization in a mouse model,5,6 endothelial cell proliferation in a retinopathy of prematurity mouse model,7 and tumor progression in a A/J Tsc2+/− C57BL/6 background mouse model of pancreatic tumor.8,9 When we initially used bevacizumab in a mouse model to treat mouse melanoma cells, we found that it slightly decreased the tumor size and number of metastases. We then performed bevacizumab experiments with human uveal melanoma cell lines. Our experiments showed that bevacizumab had much more effect on human melanoma than on mouse melanoma. We acknowledge point three of Sharma et al. and reply that we meant that 10 and 100 μg/mL of bevacizumab reduced VEGF levels by 8.51 and 15.99 pg/mL, respectively. Bevacizumab acts as a tumor suppressor by inhibiting angiogenesis and lymphangiogenesis. Lymphangiogenesis not only plays an important role in mediating immune reactions, but also facilitates tumor metastasis10. In our study, we investigated only the antiangiogensis function of bevacizumab.

Endogenous Matrix-Derived Inhibitors of Angiogenesis

Endogenous inhibitors of angiogenesis are proteins or fragments of proteins that are formed in the body, which can inhibit the angiogenic process. These molecules can be found both in the circulation and sequestered in the extracellular matrix (ECM) surrounding cells. Many matrix-derived inhibitors of angiogenesis, such as endostatin, tumstatin, canstatin and arresten, are bioactive fragments of larger ECM molecules. These substances become released upon proteolysis of the ECM and the vascular basement membrane (VBM) by enzymes of the tumor microenvironment. Although the role of matrix-derived angiogenesis inhibitors is well studied in animal models of cancer, their role in human cancers is less established. In this review we discuss the current knowledge about these molecules and their potential use as cancer therapeutics and biomarkers.

Nociceptin and its natural and specifically‐modified fragments: Structural studies

The vibrational structures of Nociceptin (FQ), its short bioactive fragments, and specifically-modified [Tyr¹]FQ (1-6), [His¹]FQ (1-6), and [His(1,4)]FQ (1-6) fragments were characterized. We showed that in the solid state, all of the aforementioned peptides except FQ adopt mainly turn and disordered secondary structures with a small contribution from an antiparallel β-sheet conformation. FQ (1-11), FQ (7-17) [His¹]FQ (1-6), and [His(1,4)]FQ (1-6) have an α-helical backbone arrangement that could also slightly influence their secondary structure. The adsorption behavior of these peptides on a colloidal silver surface in an aqueous solution (pH = ∼8.3) was investigated by means of surface-enhanced Raman scattering (SERS). All of the peptides, excluding FQ (7-17), chemisorbed on the colloidal silver surfaces through a Phe⁴ residue, which for FQ, FQ (1-11), FQ (1-6), [Tyr¹]FQ (1-6), and [His¹]FQ (1-6) lies almost flat on this surface, while for FQ (1-13) and FQ (1-13)NH₂ adopts a slightly tilted orientation with respect to the surface. The Tyr¹ residue in [Tyr¹]FQ (1-6) does not interact with the colloidal silver surface, suggesting that the Tyr¹ and Phe⁴ side chains are located on the opposite sides of the peptide backbone, which can be also true for His¹ and Phe⁴ in [His¹]FQ (1-6). The lone pair of electrons on the oxygen atom of the ionized carbonyl group of FQ (1-13) and FQ (7-17) appears to be coordinated to the colloidal silver nanoparticles, whereas in the case of the remaining peptides, it only assists in the adsorption process, similar to the --NH⁴ group. We also showed that upon adsorption, the secondary structure of these peptides is altered.

Multi-factorial modulation of IGD motogenic potential in MSF (Migration Stimulating Factor)

Migration Stimulating Factor (MSF) is a genetically truncated isoform of fibronectin (Fn). MSF is a potent stimulator of fibroblast migration, whereas full length Fn is devoid of motogenic activity. MSF and Fn contain four IGD motifs, located in the 3rd, 5th, 7th and 9th type I modules; these modules are referred to as 3FnI, 5FnI, 7FnI and 9FnI, respectively. We have previously reported that mutation of IGD motifs in modules 7FnI and 9FnI of MSF is sufficient to completely abolish the motogenic response of target adult skin fibroblasts. We now report that the IGD sequences in 3FnI and 5FnI are also capable of exhibiting motogenic activity when present within fragments of MSF. When present within 1–5FnI, these sequences require the presence of serum or vitronectin for their motogenic activity to be manifest, whereas the IGD sequences in 7FnI and 9FnI are bioactive in the absence of serum factors. All MSF and IGD-containing peptides stimulated the phosphorylation of the integrin binding protein focal adhesion kinase (FAK) but did not necessarily affect migration. These results suggest that steric hindrance determines the motogenic activity of MSF and Fn, and that both molecules contain cryptic bioactive fragments.

The Role of Complement Factor H in Age-related Macular Degeneration: A Review

Factor H is a 155 kDa sialic acid containing glycoprotein that plays an integral role in the regulation of the complement-mediated immune system that is involved in microbial defense, immune complex processing, and programmed cell death. These events take place primarily in fluid phase and on the cell surface and are particularly important in the context of distinguishing self from non-self. Activation of the complement system occurs within seconds and results in a proteolytic cascade eventually forming the membrane attack complex leading to cell lysis. Factor H protects host cells from injury resulting from unrestrained complement activation. Mutations and SNPs (single nucleotide polymorphisms) in Factor H have been implicated in a variety of human conditions including age-related macular degeneration (AMD), atypical hemolytic uremic syndrome, and membranoproliferative glomuleronephritis type II or dense deposit disease. It should not be surprising that these seemingly unrelated diseases involving mutations in Factor H may share common features. Because the immune process involves, in part, an inflammatory response and common or similar surface antigens, it is also not unexpected to observe features of inflammation, including deposition of bioactive complement fragments such as C3a and C5a, a cellular influx of immune related cells such as lymphocytes, and the potential for multiple organ involvement. We review recent developments in molecular genetics; SNPs, including Y402H; the three-dimensional structure; and mass spectroscopy of Factor H as it relates to the pathogenesis of eye disease. In addition, we discuss the concepts of molecular mimicry, sequestered or hidden antigens, and antigenic cross reactivity, and propose that AMD should not simply be considered to be an eye disease, but rather a systemic vascular disease where the eye has the ability to self regulate a local immune response. Identification of the initial event or inciting antigen has yet to be determined and will significantly advance the understanding of the pathogenesis of AMD.

The molecular biology of moenomycins: towards novel antibiotics based on inhibition of bacterial peptidoglycan glycosyltransferases

Moenomycins are phosphoglycolipid antibiotics and the only known natural product inhibitors of peptidoglycan glycosytransferases (PGTs). Techniques that would allow facile diversification of the moenomycin structure would facilitate the development of novel antibiotics, which are urgently needed in the wake of multidrug resistant bacterial infections. The cloning and initial characterization of the moenomycin biosynthetic genes has already redefined the minimal moenomycin pharmacophore and now opens the door for the biocombinatorial generation of bioactive moenomycin fragments. Here, we highlight the importance of research on the genetic mechanisms that regulate moenomycin biosynthesis and that confer moenomycin resistance to bacteria in the development of novel anti-infectives based on PGT inhibition.

Involution Is an Inflammatory Tumor Promoting Event That Increases Metastasis in Pregnancy Associated Breast Cancer.

Abstract Breast cancer diagnosed up to 6 years after a completed child-birth has an increased risk of metastatic recurrence and death, which is independent of current prognostic factors. These pregnancy-associated breast cancers (PABC) account for 45% of all young women's breast cancers and have a 10 year survival rate of 44% compared with 69% for non-PABC. After parturition or lactation, a physiologically normal tissue remodeling program is activated that mimics wound healing and inflammation, including high matrix metalloproteinase -2, -3, and -9 activities, release of bioactive fragments of FN and LN, deposition of fibrillar collagen, and increased cytokine levels. We hypothesize that the normal physiologic, but pro-inflammatory remodeling of breast during involution accounts for increased breast cancer metastasis of PABC.To demonstrate that the human breast is characterized by a pro-inflammatory microenvironment with wound-healing characteristics, formalin fixed breast biopsy tissues were obtained for women who were age-matched nulliparous, pregnant, lactating, whose breast tissue was actively involuting at the time of biopsy or who were > 5 years from a completed pregnancy. Using a novel imaging program, over 1500 images of human breast tissue have been quantitated for CD45 (common leukocyte antigen) and CD68 (macrophage lysosomal marker). CD45 and CD68 staining in the human breast demonstrates 4 times more staining in involuting compared to pregnant tissue. Further, CD45 and CD68 staining returns to baseline level in the fully regressed breast. Further characterization of the macrophages in involution demonstrates an M2 phenotype, similar to immune suppressive tumor associated macrophages. Likewise, increased collagen deposition is present with involution in comparison to the other tissue subgroups, which may help promote immune cell recruitment.To test for the impact of involution on invasion and metastasis of PABC, we have developed the MCF-10DCIS human cell line into a murine model for PABC. In this model, the cancer cells will form in situ ductal carcinoma like lesions that progress over time with loss of their myoepithelial barrier and subsequent invasion present. Orthotopic injection of the MCF-10DCIS cells into SCID mice that whose mammary glands were nulliparous versus actively involuting following timed weaning demonstrated an increased incidence of tumor of 78% involution group versus 50% in the nulliparous group. Tumor size was increased 4 fold and presence of invasive lesions increased 2 fold for the involution group. Moreover, increased lung metastasis that was independent of the primary tumor size was seen in the involuting mice compared with nulliparous controls, isolating the event of involution (independent of the potential potentiating effect of the pregnancy state on the tumor) as the driving feature for the metastatic increase.These data demonstrate for the first time that human mammary gland involution is characterized by a pro-inflammatory microenvironment, implicating physiologic tissue inflammation in the poor prognosis of PABC. Likewise, in a murine model for PABC, involution is associated with increased tumor invasion and metastasis. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 50.

Using AFM and force spectroscopy to determine pectin structure and (bio) functionality

Pectin is an integral component of non-graminaceous plant cell walls. It is believed to form an interconnected network structure independent of the cellulose-xyloglucan network structure. Pectin gels are often used as a model for the pectin network structure within the plant cell wall. Atomic force microscopy studies of calcium-induced gel precursors, and fragments released from gels, suggest that association leads to a branched fibrous structure within the gels. Enzymatic de-esterification of high-methoxyl pectin in the presence of calcium ions can induce gelation of the pectin. Thus pectin gel networks may provide a model for a self-assembled network structure within the middle lamella region of the plant cell wall. The pectin network in plant cell walls is a source of soluble and insoluble fibre. In addition to the health benefits associated with the dietary fibre aspects of pectin new health claims are emerging. Recently published in vitro and in vivo animal studies, and human studies, suggest that oral consumption of a modified form of pectin may have anti-cancer properties. These studies suggest that the modified pectin may act on a range of cancers at several stages of progression of the cancer. It has been hypothesised that this generic action is due to the modification allowing release of bioactive fragment(s) which are claimed to bind specifically to and inhibit the action of the mammalian lectin galectin 3 (Gal3). Gal3 is a key regulator of cellular homeostasis and plays important roles in several stages of cancer metastasis. Studies using force spectroscopy, flow cytometry and fluorescence microscopy suggest that the bioactive fragments of pectin may be pectin-derived galactans.

Opioid peptides derived from food proteins suppress aggregation and promote reactivation of partly unfolded stressed proteins

A new view of the opioid peptides is presented. The potential of small peptides derived from precursor food proteins, to bind to partly unfolded stressed proteins to prevent their irreversible aggregation and inactivation has been demonstrated in various in vitro test systems: dithiothreitol-induced aggregation of alpha-lactalbumin (LA), heat-induced aggregation of alcohol dehydrogenase (ADH), and aggregation and inactivation of bovine erythrocyte carbonic anhydrase (CA) in the process of its refolding after removal of stress conditions. Using dynamic light scattering (DLS), turbidimetry, fluorescence, and circular dichroism measurements protective effects of the synthetic opioid peptides: exorphin C from wheat gluten (Tyr-Pro-Ile-Ser-Leu), rubiscolin-5 from spinach ribulose-bisphosphate-carboxylase/oxygenase (Rubisco) (Tyr-Pro-Leu-Asp-Leu), and hemorphin-6 from bovine hemoglobin (Tyr-Pro-Trp-Thr-Gln-Arg) have been revealed. We have demonstrated the concentration-dependent suppression of light scattering intensity of aggregates of LA and ADH in the presence of the peptides, the population of nanoparticles with higher hydrodynamic radii being shifted to the lower ones, accompanied by an increase in the lag period of aggregation. The presence of the peptides in the refolding solution was shown to assist reactivation of CA and enhance the yield of the CA soluble protein. The results suggest that bioactive food protein fragments may be regarded as exogenous supplements to the endogenous defense mechanisms of the human organism under stress conditions.

Deletion of the Semaphorin, Sema4D, but Not Inhibition of Sema4D Shedding by ADAM17, Impairs Platelet Function and Reduces Infarct Size After Myocardial Ischemia.

Abstract 771Introduction. Platelets are activated early during the reperfusion of ischemic myocardium, potentially exacerbating the extent of ischemia/reperfusion (I/R) injury. We have recently shown (Zhu, et al., PNAS 2007) that platelets express the semaphorin family member, sema4D, as do T-cells. Sema4D is a cell surface protein whose receptors are expressed by B-cells, monocytes and endothelial cells as well as platelets. Loss of sema4D expression in mice causes a defect in signaling downstream of the platelet collagen receptor, glycoprotein (GP) VI, inhibiting platelet function in vitro and in vivo, and reducing the extent of platelet hyperresponsiveness and atherothrombosis in the setting of dyslipidemia (Zhu, et al. ATVB 2009). Because of the role played by platelets, leukocytes and endothelial cells in reperfusion injury, here we asked whether loss of sema4D expression can also protect the heart, reducing the extent of damage following temporary ischemia. Methods. The left anterior descending coronary artery of anaesthetized mice was ligated for 45 min. After reperfusion for 48 hours, the mice were re-anesthetized and perfused with 2,3,5-triphenyltetrazolium chloride to measure the area of infarction. Fluorescent microspheres were used to delineate the area at risk. Comparisons were made between sema4D(−/−) and wild type mice produced by breeding sema4D(+/−) heterozygotes. Results. Although there was no difference between the sema4D(−/−) and WT mice with respect to either heart size or area at risk, we observed a substantial (57%) decrease in infarct size in the sema4D(−/−) mice expressed as a fraction of the area at risk (N=7-9, p<0.005). Since sema4D is shed from the surface of activated platelets and T-cells by the metalloprotease, ADAM17, producing a large bioactive fragment, we next asked whether the protection against ischemia/reperfusion injury conferred by the sema4D knockout is due to the loss of cell-associated or soluble sema4D. Chimeric mice were produced in which hematopoiesis was reconstituted in irradiated sema4D(+/+) mice using fetal liver cells from mouse embryos that lack functional ADAM17. This produces mice in which sema4D is expressed as usual in the hematopoietic lineages, but unable to be shed. Chimerism, inhibition of sema4D shedding and recovery of normal cell counts were confirmed after transplantation. The ischemia/reperfusion studies were repeated comparing chimeras reconstituted with ADAM17-deficient and ADAM17-replete fetal liver cells. In contrast to the sema4D knockout, the extent of infarction was the same whether or not ADAM17 was functional and sema4D was shed. Conclusions. Although the role of sema4D and its receptors have been studied most extensively in the context of T-cell interactions with B-cells, our previous studies have made a case for the involvement of sema4D in platelet:platelet and platelet:endothelial cell interactions. We now show for the first time that 1) loss of sema4D expression in mice confers protection against ischemia/reperfusion injury in the myocardium, and 2) preventing the formation of soluble, bioactive sema4D is insufficient to recapitulate this effect. Since sema4D and its receptors are expressed on more than just platelets, it cannot be concluded that the observed protection in the knockout is solely due to the absence of platelet sema4D. However, experience with other knockouts that reduce platelet function suggests that the defects that we have observed in sema4D(−/−) platelet function are likely to contribute. Regardless of whether expression on platelets is entirely or only partly responsible for the observed phenotype, sema4D is an interesting target for therapeutic intervention. Disclosures:No relevant conflicts of interest to declare.

ADAMTS Metalloproteases Generate Active Versican Fragments that Regulate Interdigital Web Regression

We show that combinatorial mouse alleles for the secreted metalloproteases Adamts5, Adamts20 (bt), and Adamts9 result in fully penetrant soft-tissue syndactyly. Interdigital webs in Adamts5(-/-);bt/bt mice had reduced apoptosis and decreased cleavage of the proteoglycan versican; however, the BMP-FGF axis, which regulates interdigital apoptosis was unaffected. BMP4 induced apoptosis, but without concomitant versican proteolysis. Haploinsufficiency of either Vcan or Fbln1, a cofactor for versican processing by ADAMTS5, led to highly penetrant syndactyly in bt mice, suggesting that cleaved versican was essential for web regression. The local application of an aminoterminal versican fragment corresponding to ADAMTS-processed versican, induced cell death in Adamts5(-/-);bt/bt webs. Thus, ADAMTS proteases cooperatively maintain versican proteolysis above a required threshold to create a permissive environment for apoptosis. The data highlight the developmental significance of proteolytic action on the ECM, not only as a clearance mechanism, but also as a means to generate bioactive versican fragments.

Role of the extracellular matrix in lymphocyte migration

The extracellular matrix (ECM) exists in various biochemical and structural forms that can act either as a barrier to migrating leukocytes, in the case of basement membranes, or provide a physical scaffold supporting or guiding migration (interstitial matrix). This review focuses on basement membranes and our current knowledge of the way that leukocytes transmigrate this protein barrier, with emphasis on T lymphocytes. Recent data suggest that the classical concept of cell-matrix adhesion requires revision with respect to leukocyte-ECM interactions. Whereas specific receptors may be required for leukocyte recognition of ECM molecules or three-dimensional structural domains, the role of adhesion in migration as perceived from the traditional studies of adherent cell-ECM interactions is less clear. Further, the indirect effects of ECM such as the binding and presentation of cytokines or chemotactic factors may more profoundly influence the directed migration of normally non-adherent leukocytes than the migration of adherent cells such as epithelial cells or fibroblasts. Proteases (in particular matrix metalloproteinases) released at sites of inflammation can selectively process ECM, cell surface molecules or soluble factors, which may result in the release of bioactive fragments that can function as chemoattractants for different leukocyte subsets or may modulate the activity/function of resident mesenchymal and immune cells. Current findings suggest that different leukocyte types employ different mechanisms to migrate across or through the ECM; this might be determined by the composition and organization of the ECM itself.

Analysis of Domains in Selected Plant and Animal Food Proteins - Precursors of Biologically Active Peptides - In Silico Approach

In silico methods are useful tool in protein structure-functional relationships analysis. BIOPEP and InterPro databases were applied to analyze the presence of bioactive fragments in the domains occurring in the sequences representing the major groups of proteins. Domains found in the proteins analyzed had mostly transporting (bovine β-lactoglobulin), immunoglobulin-like (chicken connectin), alpha-amylase inhibitor (a/β-wheat gliadin), calcium binding (chicken myosin) functions, or allowed straightly to assign the protein to an appropriate superfamily (bovine casein). It confirmed the thesis about the existence of the functional relations between the structure (sequence) and the domains with identified conformation. Amongst the domains present in the protein sequences we revealed the presence of fragments with the activities: antihypertensive, opioid, dipeptidylpeptidase IV inhibitors, immunomodulating, and neuropeptides. In the chicken connectin within the immunoglobulin-like domain we found immunomodulating fragments. InterPro analysis did not reveal the existence of any domains in a soybean globulin. It can be explained by the lack of the key structure information helpful in the defining the structure-function relationships. As the number of information in the applied databases will continue to increase we can expect to find stronger relationships between bioactivity of fragments encrypted in proteins and the functionality of domains. This might allow in the future to find evolutionary similarity between different origin food proteins - sources of bioactive peptides.

Multiple Roles of the Extracellular Matrix in Inflammation

Extracellular matrix (ECM) provides a physical scaffold for cells but also provides specific molecular and spatial information that influences cell proliferation, differentiation and apoptosis. This review addresses the multiple roles of ECM in inflammatory responses, in particular in leukocyte extravasation at sites of inflammation, and the potential of exploiting such cell-ECM interactions to interfere with defined steps in the inflammatory process. In the course of an inflammation leukocytes not only have to penetrate the vascular endothelial cell monolayer, but also the underlying endothelial cell basement membrane and invade the interstitial matrix of the stroma to reach the site of inflammation. The endothelial cell basement membrane may directly influence leukocyte recruitment to the inflammed tissue by providing differential signals resulting from its spatial and molecular composition, or indirectly by its potential to bind and present cytokines or chemotactic factors. Proteases (in particular matrix metalloproteinases (MMP)) released at sites of inflammation selectively process ECM and cell surface molecules, which may result in the release of bioactive fragments that may function as chemoattractants for different leukocytes subsets or modulate the activity/ function of resident mesenchymal and immune cells. In addition, MMPs have been shown to process chemokines modulating their chemoattractant properties. To be able to mimic or inhibit some of the ECM functions or proteolytic events that occur during inflammation, through the use of specific protein fragments, would provide a means by which the inflammatory process could be manipulated, an area however that remains largely unexplored.

Common wound healing signature in murine and human involuting breast, implications for pregnancy-associated breast cancer.

Abstract Abstract #1049 Full term pregnancy increases breast cancer risk for up to ten years after childbirth and breast cancer diagnosed during this time has been referred to as pregnancy associated breast cancer (PABC). PABC patients have high rates of metastasis, which is independent of known prognostic factors. Following parturition or lactation, during involution, the breast returns to its pre-pregnant state and in the rat model, we found mammary gland involution employs tissue remodeling programs activated during wound healing and inflammation including high matrix metalloproteinase -2, -3, and -9 activities, release of bioactive fragments of FN and LN, deposition of fibrillar collagen, and increased cytokine levels, including TGFβ1, TGFβ3 and CTGF. We hypothesize that this physiologically normal, but pro-inflammatory remodeling of the gland accounts for the high rate of metastases seen in PABC.&amp;#x2028; To further define involution features common to wound healing that may account for the metastasis promoting attributes of PABC, the question of whether macrophages are recruited to the involuting gland was investigated in both rat mammary and human breast tissue. Using the macrophage marker CD68, a lysosomal associated protein, the number of CD68 positive cells in rat mammary glands increased more than 6 fold during involution. Involution-associated macrophages were identified as subtype M2-like by arginase-1 expression and lack of subtype M1-marker iNOS expression. To address the question of whether breast involution in women also is characterized by a wound-healing signature, formalin fixed breast biopsy tissues were obtained for women who were either pregnant, lactating, or whose breast tissue was actively involuting at the time of biopsy. Using a novel computer assisted imaging program, over 200 images of human breast tissue have been quantitated for CD68. CD68 staining in the human breast followed the same trend as in rat, with 5 times more staining in involuting compared to pregnant or lactating tissue. Total leukocyte number, as assessed by CD45 antibody, and oncofetal ECM protein tenascin-C staining were also increased in involuting human breast tissue. With respect to these three stromal markers, involuting human breast tissue was more similar to breast cancer stroma (N=50 images) than stroma from breasts of nulliparous women (N=30 images). These data demonstrate for the first time that human mammary gland involution is characterized by a pro-inflammatory microenvironment, implicating physiologic tissue inflammation in the poor prognosis of PABC.&amp;#x2028; In a newly developed animal model for PABC, MCF10DCIS cells were injected orthotopically into SCID mice whose mammary glands were either quiescent (virgin mice) or actively remodeling following weaning (involuting mice). Tumor incidence was 78% in involuting and 50% in virgin mice. Further, tumor multiplicity, size and DCIS conversion to locally invasive lesions were significantly higher in the involuting mice than control virgin mice. This model is being utilized for mechanistic and chemopreventive studies of PABC. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1049.

Cell surface heparan sulfate released by heparanase promotes melanoma cell migration and angiogenesis

Heparan sulfate (HS) proteoglycans are essential components of the cell-surface and extracellular matrix (ECM) which provide structural integrity and act as storage depots for growth factors and chemokines, through their HS side chains. Heparanase (HPSE) is the only mammalian endoglycosidase known that cleaves HS, thus contributing to matrix degradation and cell invasion. The enzyme acts as an endo-beta-D-glucuronidase resulting in HS fragments of discrete molecular weight size. Cell-surface HS is known to inhibit or stimulate tumorigenesis depending upon size and composition. We hypothesized that HPSE contributes to melanoma metastasis by generating bioactive HS from the cell-surface to facilitate biological activities of tumor cells as well as tumor microenvironment. We removed cell-surface HS from melanoma (B16B15b) by HPSE treatment and resulting fragments were isolated. Purified cell-surface HS stimulated in vitro B16B15b cell migration but not proliferation, and importantly, enhanced in vivo angiogenesis. Furthermore, melanoma cell-surface HS did not affect in vitro endothelioma cell (b.End3) migration. Our results provide direct evidence that, in addition to remodeling ECM and releasing growth factors and chemokines, HPSE contributes to aggressive phenotype of melanoma by releasing bioactive cell-surface HS fragments which can stimulate melanoma cell migration in vitro and angiogenesis in vivo.

Caveolin-1, transforming growth factor-β receptor internalization, and the pathogenesis of systemic sclerosis

To review the scientific literature supporting the participation of caveolin-1 in the pathogenesis of tissue fibrosis and the notion that modulation of the caveolin-1 pathway may represent a novel treatment for systemic sclerosis and other fibrotic diseases. Caveolin-1 plays an important role in the regulation of transforming growth factor-beta (TGF-beta) signaling owing to its participation in TGF-beta receptor internalization. TGF-beta receptor internalized through caveolin-1 lipid rafts undergoes rapid degradation, effectively decreasing TGF-beta signaling. Studies have shown that caveolin-1 knockdown in vitro markedly increased collagen gene expression in normal human lung fibroblasts. Caveolin-1 was reduced in affected systemic sclerosis lungs and skin and in idiopathic pulmonary fibrosis lung tissues and fibroblasts. Increasing caveolin-1 expression markedly improved bleomycin-induced pulmonary fibrosis. Restoration of caveolin bioavailability employing penetratin, a cell-permeable peptide carrier for a bioactive caveolin-1 fragment, abrogated TGF-beta activation of cultured human dermal fibroblasts. Systemic administration of penetratin-caveolin-1 peptide to mice with bleomycin-induced lung fibrosis reduced fibrosis. Caveolin-1 plays an important role in the regulation of TGF-beta signaling and participates in the pathogenesis of systemic sclerosis and idiopathic pulmonary fibrosis. Restoration of caveolin function employing active caveolin-1 fragments coupled to cell-permeable carrier peptides may represent a novel approach for their treatment.

Recognition of galactan components of pectin by galectin‐3

It has been reported that modified forms of pectin possess anticancer activity. To account for this bioactivity, it has been proposed that fragments of pectin molecules can act by binding to and inhibiting the various roles of the mammalian protein galectin 3 (Gal3) in cancer progression and metastasis. Despite this clear molecular hypothesis and evidence for the bioactivity of modified pectin, the structural origins of the "bioactive fragments" of pectin molecules are currently ill defined. By using a combination of fluorescence microscopy, flow cytometry, and force spectroscopy, it has been possible to demonstrate, for the first time, specific binding of a pectin galactan to the recombinant form of human Gal3. Present studies suggest that bioactivity resides in the neutral sugar side chains of pectin polysaccharides and that these components could be isolated and modified to optimize bioactivity.