Identification of a Novel Structural Class of HV1 Inhibitors by Structure-Based Virtual Screening.

The human voltage-gated proton channel, hHV1, is highly expressed in various cell types including macrophages, B lymphocytes, microglia, sperm cells and also in various cancer cells. Overexpression of HV1 has been shown to promote tumor formation by highly metastatic cancer cells, and has been associated with neuroinflammatory diseases, immune response disorders and infertility, suggesting a potential use of hHV1 inhibitors in numerous therapeutic areas. To identify compounds targeting this channel, we performed a structure-based virtual screening on an open structure of the human HV1 channel. Twenty selected virtual screening hits were tested on Chinese hamster ovary (CHO) cells transiently expressing hHV1, with compound 13 showing strong block of the proton current with an IC50 value of 8.5 µM. Biological evaluation of twenty-three additional analogs of 13 led to the discovery of six other compounds that blocked the proton current by more than 50% at 50 µM concentration. This allowed for an investigation of structure-activity relationships. The antiproliferative activity of the selected promising hHV1 inhibitors was investigated in the cell lines MDA-MB-231 and THP-1, where compound 13 inhibited growth with an IC50 value of 9.0 and 8.1 µM, respectively. The identification of a new structural class of HV1 inhibitors contributes to our understanding of the structural requirements for inhibition of this ion channel and opens up the possibility of investigating the role of HV1 inhibitors in various pathological conditions and in cancer therapy.

Influence of N-arylsulfonamido d-valine N-substituents on the selectivity and potency of matrix metalloproteinase inhibitors.

To develop matrix metalloproteinase inhibitors (MMPIs) for both therapy and medicinal imaging by fluorescence-based techniques or positron-emission tomography (PET), a small library of eighteen N-substituted N-arylsulfonamido d-valines were synthesized and their potency to inhibit two gelatinases (MMP-2, and MMP-9), two collagenases (MMP-8, and MMP-13) and macrophage elastase (MMP-12) was determined in a Structure-Activity-Relation study with ({4-[3-(5-methylthiophen-2-yl)-1,2,4-oxadiazol-5-yl]phenyl}sulfonyl)-d-valine (1) as a lead. All compounds were shown to be more potent MMP-2/-9 inhibitors (nanomolar range) compared to other tested MMPs. This is a remarkable result considering that a carboxylic acid group is the zinc binding moiety. The compound with a terminal fluoropropyltriazole group at the furan ring (P1' substituent) was only four times less potent in inhibiting MMP-2 activity than the lead compound 1, making this compound a promising probe for PET application (after using a prosthetic group approach to introduce fluorine-18). Compounds with a TEG spacer and a terminal azide or even a fluorescein moiety at the sulfonylamide N atom (P2' substituent) were almost as active as the lead structure 1, making the latter derivative a suitable fluorescence imaging tool.

The Voltage-Gated Hv1 H+ Channel Is Expressed in Tumor-Infiltrating Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSCs) are key determinants of the immunosuppressive microenvironment in tumors. As ion channels play key roles in the physiology/pathophysiology of immune cells, we aimed at studying the ion channel repertoire in tumor-derived polymorphonuclear (PMN-MDSC) and monocytic (Mo-MDSC) MDSCs. Subcutaneous tumors in mice were induced by the Lewis lung carcinoma cell line (LLC). The presence of PMN-MDSC (CD11b+/Ly6G+) and Mo-MDSCs (CD11b+/Ly6C+) in the tumor tissue was confirmed using immunofluorescence microscopy and cells were identified as CD11b+/Ly6G+ PMN-MDSCs and CD11b+/Ly6C+/F4/80-/MHCII- Mo-MDSCs using flow cytometry and sorting. The majority of the myeloid cells infiltrating the LLC tumors were PMN-MDSC (~60%) as compared to ~10% being Mo-MDSCs. We showed that PMN- and Mo-MDSCs express the Hv1 H+ channel both at the mRNA and at the protein level and that the biophysical and pharmacological properties of the whole-cell currents recapitulate the hallmarks of Hv1 currents: ~40 mV shift in the activation threshold of the current per unit change in the extracellular pH, high H+ selectivity, and sensitivity to the Hv1 inhibitor ClGBI. As MDSCs exert immunosuppression mainly by producing reactive oxygen species which is coupled to Hv1-mediated H+ currents, Hv1 might be an attractive target for inhibition of MDSCs in tumors.

Identification and characterisation of tertiary lymphoid organs in human type 1 diabetes.

AIMS/HYPOTHESIS: We and others previously reported the presence of tertiary lymphoid organs (TLOs) in the pancreas of NOD mice, where they play a role in the development of type 1 diabetes. Our aims here are to investigate whether TLOs are present in the pancreas of individuals with type 1 diabetes and to characterise their distinctive features, in comparison with TLOs present in NOD mouse pancreases, in order to interpret their functional significance. METHODS: Using immunofluorescence confocal microscopy, we examined the extracellular matrix (ECM) and cellular constituents of pancreatic TLOs from individuals with ongoing islet autoimmunity in three distinct clinical settings of type 1 diabetes: at risk of diabetes; at/after diagnosis; and in the transplanted pancreas with recurrent diabetes. Comparisons were made with TLOs from 14-week-old NOD mice, which contain islets exhibiting mild to heavy leucocyte infiltration. We determined the frequency of the TLOs in human type 1diabetes with insulitis and investigated the presence of TLOs in relation to age of onset, disease duration and disease severity. RESULTS: TLOs were identified in preclinical and clinical settings of human type 1 diabetes. The main characteristics of these TLOs, including the cellular and ECM composition of reticular fibres (RFs), the presence of high endothelial venules and immune cell subtypes detected, were similar to those observed for TLOs from NOD mouse pancreases. Among 21 donors with clinical type 1 diabetes who exhibited insulitis, 12 had TLOs and had developed disease at younger age compared with those lacking TLOs. Compartmentalised TLOs with distinct T cell and B cell zones were detected in donors with short disease duration. Overall, TLOs were mainly associated with insulin-containing islets and their frequency decreased with increasing severity of beta cell loss. Parallel studies in NOD mice further revealed some differences in so far as regulatory T cells were essentially absent from human pancreatic TLOs and CCL21 was not associated with RFs. CONCLUSIONS/INTERPRETATION: We demonstrate a novel feature of pancreas pathology in type 1 diabetes. TLOs represent a potential site of autoreactive effector T cell generation in islet autoimmunity and our data from mouse and human tissues suggest that they disappear once the destructive process has run its course. Thus, TLOs may be important for type 1 diabetes progression.

Blockade of MCAM/CD146 impedes CNS infiltration of T cells over the choroid plexus.

BACKGROUND: Very late antigen 4 (VLA-4; integrin α4ß1) is critical for transmigration of T helper (TH) 1 cells into the central nervous system (CNS) under inflammatory conditions such as multiple sclerosis (MS). We have previously shown that VLA-4 and melanoma cell adhesion molecule (MCAM) are important for trans-endothelial migration of human TH17 cells in vitro and here investigate their contribution to pathogenic CNS inflammation. METHODS: Antibody blockade of VLA-4 and MCAM is assessed in murine models of CNS inflammation in conjunction with conditional ablation of α4-integrin expression in T cells. Effects of VLA-4 and MCAM blockade on lymphocyte migration are further investigated in the human system via in vitro T cell transmigration assays. RESULTS: Compared to the broad effects of VLA-4 blockade on encephalitogenic T cell migration over endothelial barriers, MCAM blockade impeded encephalitogenic T cell migration in murine models of MS that especially depend on CNS migration across the choroid plexus (CP). In transgenic mice lacking T cell α4-integrin expression (CD4::Itga4-/-), MCAM blockade delayed disease onset. Migration of MCAM-expressing T cells through the CP into the CNS was restricted, where laminin 411 (composed of α4, ß1, γ1 chains), the proposed major ligand of MCAM, is detected in the endothelial basement membranes of murine CP tissue. This finding was translated to the human system; blockade of MCAM with a therapeutic antibody reduced in vitro transmigration of MCAM-expressing T cells across a human fibroblast-derived extracellular matrix layer and a brain-derived endothelial monolayer, both expressing laminin α4. Laminin α4 was further detected in situ in CP endothelial-basement membranes in MS patients' brain tissue. CONCLUSIONS: Our findings suggest that MCAM-laminin 411 interactions facilitate trans-endothelial migration of MCAM-expressing T cells into the CNS, which seems to be highly relevant to migration via the CP and to potential future clinical applications in neuroinflammatory disorders.

Staphylococcus aureus requires less virulence to establish an infection in diabetic hosts.

Staphylococcus aureus is the most frequent pathogen causing diabetic foot infections. Here, we investigated the degree of bacterial virulence required to establish invasive tissue infections in diabetic organisms. Staphylococcal isolates from diabetic and non-diabetic foot ulcers were tested for their virulence in in vitro functional assays of host cell invasion and cytotoxicity. Isolates from diabetes mellitus type I/II patients exhibited less virulence than isolates from non-diabetic patients, but were nevertheless able to establish severe infections. In some cases, non-invasive isolates were detected deep within diabetic wounds, even though the strains were non-pathogenic in cell culture models. Testing of defined isolates in murine footpad injection models revealed that both low- and high-virulent bacterial strains persisted in higher numbers in diabetic compared to non-diabetic hosts, suggesting that hyperglycemia favors bacterial survival. Additionally, the bacterial load was higher in NOD mice, which have a compromised immune system, compared to C57Bl/6 mice. Our results reveal that high as well as low-virulent staphylococcal strains are able to cause soft tissue infections and to persist in diabetic humans and mice, suggesting a reason for the frequent and endangering infections in patients with diabetes.

New in Vivo Compatible Matrix Metalloproteinase (MMP)-2 and MMP-9 Inhibitors.

Matrix metalloproteinases (MMPs) are emerging as pivotal fine-tuners of cell function in tissue homeostasis and in various pathologies, in particular inflammation. In vivo monitoring of the activity of specific MMPs, therefore, provides high potential for assessing disease progression and tissue function, and manipulation of MMP activity in tissues and whole organisms may further provide a mode of controlling pathological processes. We describe here the synthesis of novel fluorinated and nonfluorinated analogues of a secondary sulfonamide-based lead structure, compound 2, and test their efficacy as in vivo inhibitors and tracers of the gelatinases, MMP-2 and MMP-9. Using a murine neuroinflammatory model, we show that compound 2 is a highly effective in vivo inhibitor of both MMP-2 and MMP-9 activity with little or no adverse effects even after long-term daily oral administration. A fluorescein-labeled derivative compound 17 shows direct binding to activated gelatinases surrounding inflammatory cuffs in the neuroinflammation model and to pancreatic ß-cells in the islets of Langerhans, colocalizing with MMP-2 and MMP-9 activity as detected using in situ zymography techniques. These results demonstrate that compound 2 derivatives have potential as in vivo imaging tools and for future development for specific MMP-2 versus MMP-9 probes. Our chemical modifications mainly target the residues directed toward the S1' and S2' pockets and, thereby, provide new information on the structure-activity relationships of this inhibitor type.

Extracellular deposition of matrilin-2 controls the timing of the myogenic program during muscle regeneration.

Here, we identify a role for the matrilin-2 (Matn2) extracellular matrix protein in controlling the early stages of myogenic differentiation. We observed Matn2 deposition around proliferating, differentiating and fusing myoblasts in culture and during muscle regeneration in vivo. Silencing of Matn2 delayed the expression of the Cdk inhibitor p21 and of the myogenic genes Nfix, MyoD and Myog, explaining the retarded cell cycle exit and myoblast differentiation. Rescue of Matn2 expression restored differentiation and the expression of p21 and of the myogenic genes. TGF-ß1 inhibited myogenic differentiation at least in part by repressing Matn2 expression, which inhibited the onset of a positive-feedback loop whereby Matn2 and Nfix activate the expression of one another and activate myoblast differentiation. In vivo, myoblast cell cycle arrest and muscle regeneration was delayed in Matn2(-/-) relative to wild-type mice. The expression levels of Trf3 and myogenic genes were robustly reduced in Matn2(-/-) fetal limbs and in differentiating primary myoblast cultures, establishing Matn2 as a key modulator of the regulatory cascade that initiates terminal myogenic differentiation. Our data thus identify Matn2 as a crucial component of a genetic switch that modulates the onset of tissue repair.

Extracellular matrix components in the pathogenesis of type 1 diabetes.

Type 1 diabetes (T1D) results from progressive immune cell-mediated destruction of pancreatic ß cells. As immune cells migrate into the islets, they pass through the extracellular matrix (ECM). This ECM is composed of different macromolecules localized to different compartments within and surrounding islets; however, the involvement of this ECM in the development of human T1D is not well understood. Here, we summarize our recent findings from human and mouse studies illustrating how specific components of the islet ECM that constitute basement membranes and interstitial matrix of the islets, and surprisingly, the intracellular composition of islet ß cells themselves, are significantly altered during the pathogenesis of T1D. Our focus is on the ECM molecules laminins, collagens, heparan sulfate/heparan sulfate proteoglycans, and hyaluronan, as well as on the enzymes that degrade these ECM components. We propose that islet and lymphoid tissue ECM composition and organization are critical to promoting immune cell activation, islet invasion, and destruction of islet ß cells in T1D.

CD4(+) type II NKT cells mediate ICOS and programmed death-1-dependent regulation of type 1 diabetes.

Type 1 diabetes (T1D) is a chronic autoimmune disease that results from T cell-mediated destruction of pancreatic ß cells. CD1d-restricted NKT lymphocytes have the ability to regulate immunity, including autoimmunity. We previously demonstrated that CD1d-restricted type II NKT cells, which carry diverse TCRs, prevented T1D in the NOD mouse model for the human disease. In this study, we show that CD4(+) 24αß type II NKT cells, but not CD4/CD8 double-negative NKT cells, were sufficient to downregulate diabetogenic CD4(+) BDC2.5 NOD T cells in adoptive transfer experiments. CD4(+) 24αß NKT cells exhibited a memory phenotype including high ICOS expression, increased cytokine production, and limited display of NK cell markers, compared with double-negative 24αß NKT cells. Blocking of ICOS or the programmed death-1/programmed death ligand 1 pathway was shown to abolish the regulation that occurred in the pancreas draining lymph nodes. To our knowledge, these results provide for the first time cellular and molecular information on how type II CD1d-restricted NKT cells regulate T1D.

New 'kids' on the voltage-gated proton channel block.

So far one gene for Hv1 has been detected in studied species. The work presented by Chaves et al. in The FEBS Journal reported an 'Unexpected expansion of the voltage-gated proton channel family'. They searched for proton channel candidates and found three sequences in the genome of Aplysia californica (Ac), which were named AcHv1, AcHv2 and AcHv3. Based on electrophysiological experiments, AcHv1 and AcHv2 are voltage-gated channels. While AcHv1 behaves like Hv1 in other species, that is, it is voltage and pH-dependent, it can be inhibited by zinc and conducts protons outwardly, AcHv2 conducts protons inwards at symmetrical pH. AcHv3 constantly leaks protons, and its C-terminal part contains several cytoplasmic retention motifs. Through carefully designed and carried out electrophysiological experiments, Chaves et al. determined the biophysical parameters of all three proton channels, such as the voltage and the pH dependence, the threshold-voltage, the gating charge and the time constants of activation and inactivation. Comment on: https://doi.org/10.1111/febs.16617.

5-Chloro-2-Guanidinobenzimidazole (ClGBI) Is a Non-Selective Inhibitor of the Human HV1 Channel.

5-chloro-2-guanidinobenzimidazole (ClGBI), a small-molecule guanidine derivative, is a known effective inhibitor of the voltage-gated proton (H+) channel (HV1, Kd ≈ 26 µM) and is widely used both in ion channel research and functional biological assays. However, a comprehensive study of its ion channel selectivity determined by electrophysiological methods has not been published yet. The lack of selectivity may lead to incorrect conclusions regarding the role of hHv1 in physiological or pathophysiological responses in vitro and in vivo. We have found that ClGBI inhibits the proliferation of lymphocytes, which absolutely requires the functioning of the KV1.3 channel. We, therefore, tested ClGBI directly on hKV1.3 using a whole-cell patch clamp and found an inhibitory effect similar in magnitude to that seen on hHV1 (Kd ≈ 72 µM). We then further investigated ClGBI selectivity on the hKV1.1, hKV1.4-IR, hKV1.5, hKV10.1, hKV11.1, hKCa3.1, hNaV1.4, and hNaV1.5 channels. Our results show that, besides HV1 and KV1.3, all other off-target channels were inhibited by ClGBI, with Kd values ranging from 12 to 894 µM. Based on our comprehensive data, ClGBI has to be considered a non-selective hHV1 inhibitor; thus, experiments aiming at elucidating the significance of these channels in physiological responses have to be carefully evaluated.

Glycosaminoglycan mimicry by COAM reduces melanoma growth through chemokine induction and function.

Chlorite-oxidized oxyamylose (COAM), a glycosaminoglycan mimetic and potent antiviral agent, provided significant growth reduction of syngeneic murine B16-F1 melanoma tumors. A single early dose (100 µg, into the site of tumor cell inoculation) was sufficient to establish a persistent effect over 17 days (resected tumor volume of 78.3 mm(3) in COAM-treated mice compared to 755.2 mm(3) in the control cohort, i.e., 89.6% reduction of tumor volumes). COAM was a much better antitumoral agent than the polyanionic glycosaminoglycan heparin. COAM retained its antitumoral effect in lymphopenic mice, reinforcing the idea of myeloid cell involvement. Massive recruitment of myeloid cells into dermal air pouches in response to COAM and their increased presence in early-treated tumors indicated that mainly CD11b(+) GR-1(+) myeloid cells were attracted by COAM to exert antitumoral effects. Leukocyte chemotaxis was mediated by the chemokine system through the induction in B16-F1 cells of mouse granulocyte chemotactic protein-2/CXCL6 upon COAM treatment. Thus, COAM constitutes a novel tool to study the role of innate immune cells in the initial stages of tumor development and an example that innate immunostimulating glycosaminoglycan mimicry may be exploited therapeutically.

Sialoadhesin-positive macrophages bind regulatory T cells, negatively controlling their expansion and autoimmune disease progression.

An important regulatory suppressive function in autoimmune and other inflammatory processes has been ascribed to CD4(+)Foxp3(+) regulatory T cells (Tregs), which requires direct cell-cell communication between Tregs, effector T cells, and APCs. However, the molecular basis for these interactions has not yet been clarified. We show here that sialoadhesin (Sn), the prototype of the siglec family of sialic acid-binding transmembrane proteins, expressed by resident and activated tissue-infiltrating macrophages, directly binds to Tregs, negatively regulating their expansion in an animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). In this model, macrophages infiltrate the CNS exhibiting tissue-destructing and demyelinating activity, leading to MS-like symptoms. We show here that severity of EAE symptoms is reduced in Sn knockout (KO) mice compared with wild-type littermates due to an up-regulation of CD4(+)Foxp3(+) Treg lymphocytes. Through the use of a Sn fusion protein, Tregs were shown to express substantial amounts of Sn ligand on their cell surface, and direct interaction of Sn(+) macrophages with Tregs specifically inhibited Treg but not effector T lymphocyte proliferation. Conversely, blocking of Sn on macrophages by Sn-specific Abs resulted in elevated proliferation of Tregs. Data indicate that Sn(+) macrophages regulate Treg homeostasis which subsequently influences EAE progression. We propose a new direct cell-cell interaction-based mechanism regulating the expansion of the Tregs during the immune response, representing a "dialogue" between Sn(+) macrophages and Sn-accessible sialic acid residues on Treg lymphocytes.

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.

CCL21 Expression in ß-Cells Induces Antigen-Expressing Stromal Cell Networks in the Pancreas and Prevents Autoimmune Diabetes in Mice.

Tumors induce tolerance toward their antigens by producing the chemokine CCL21, leading to the formation of tertiary lymphoid organs (TLOs). Ins2-CCL21 transgenic, nonobese diabetic (NOD) mice express CCL21 in pancreatic ß-cells and do not develop autoimmune diabetes. We investigated by which mechanisms CCL21 expression prevented diabetes. Ins2-CCL21 mice develop TLOs by 4 weeks of age, consisting of naive CD4+ T cells compartmentalized within networks of CD45-gp38+CD31- fibroblastic reticular cell (FRC)-like cells. Importantly, 12-week-old Ins2-CCL21 TLOs contained FRC-like cells with higher contractility, regulatory, and anti-inflammatory properties and enhanced expression of ß-cell autoantigens compared with nontransgenic NOD TLOs found in inflamed islets. Consistently, transgenic mice harbored fewer autoreactive T cells and a higher proportion of regulatory T cells in the islets. Using adoptive transfer and islet transplantation models, we demonstrate that TLO formation in Ins2-CCL21 transgenic islets is critical for the regulation of autoimmunity, and although the effect is systemic, the induction is mediated locally likely by lymphocyte trafficking through TLOs. Overall, our findings suggest that CCL21 promotes TLOs that differ from inflammatory TLOs found in type 1 diabetic islets in that they resemble lymph nodes, contain FRC-like cells expressing ß-cell autoantigens, and are able to induce systemic and antigen-specific tolerance leading to diabetes prevention.

Expression of matrilin-2 in liver cirrhosis and hepatocellular carcinoma.

The recently described matrilin protein family is part of the extracellular matrix, their pathophysiological role as well as distribution in liver diseases, however, have not yet been studied. Considering that matrilins have been found to play role in cell growth and tissue remodeling, their possible involvement in carcinogenesis has been raised. The main objective of this study was to investigate the changes in matrilin-2 expression which is one of the main components of basement membranes. Thirty-five cases of surgically resected hepatocellular carcinomas, 35 corresponding surrounding liver tissues and 10 normal liver samples were used for the study. In 15 of 35 cases the tumor developed on the basis of cirrhosis. Matrilin-2 protein expression was detected in normal liver around bile ducts, portal blood vessels, while sinusoids were negative by immunohistochemistry. Cirrhotic surrounding tissue showed intensive matrilin-2 staining along the sinusoids. Tumorous neovasculature was found strongly positive by immunohistochemistry. No differences, however, were detected by morphometry regarding the amount of protein expression based on the grade of hepatocellular carcinomas. Real-time RT-PCR did not show significant differences in matrilin-2 mRNA expression between normal, cirrhotic and tumor samples. This suggests posttranslational modification of matrilin-2 manifesting in altered distribution in liver fibrosis. Our data indicate that matrilin-2 is a novel basement membrane component in the liver, which is synthetised during sinusoidal "capillarization" in cirrhosis and in hepatocellular carcinoma. This is the first report to describe the expression and distribution of matrilin-2 in human normal and cirrhotic liver as well as in hepatocellular carcinoma.

Bone marrow laminins influence hematopoietic stem and progenitor cell cycling and homing to the bone marrow.

Hematopoietic stem and progenitor cell (HSPC) functions are regulated by a specialized microenvironment in the bone marrow - the hematopoietic stem cell niche - of which the extracellular matrix (ECM) is an integral component. We describe here the localization of ECM molecules, in particular the laminin α4, α3 and α5 containing isoforms in the bone marrow. Laminin 421 (composed of laminin α4, ß2, γ1 chains) is identified as a major component of the bone marrow ECM, occurring abundantly surrounding venous sinuses and in a specialized reticular fiber network of the intersinusoidal spaces of murine bone marrow (BM) in close association with HSPC. Bone marrow from Lama4-/- mice is significantly less efficient in reconstituting the hematopoietic system of irradiated wildtype (WT) recipients in competitive bone marrow transplantation assays and shows reduced colony formation in vitro. This is partially due to retention of Lin-c-kit+Sca-1+CD48- long-term and short-term hematopoietic stem cells (LT-HSC/ST-HSC) in the G0 phase of the cell cycle in Lama4-/- bone marrow and hence a more quiescent phenotype. In addition, the extravasation of WT BM cells into Lama4-/- bone marrow is impaired, influencing the recirculation of HSPC. Our data suggest that these effects are mediated by a compensatory expression of laminin α5 containing isoforms (laminin 521/522) in Lama4-/- bone marrow. Collectively, these intrinsic and extrinsic effects lead to reduced HSPC numbers in Lama4-/- bone marrow and reduced hematopoietic potential.

Expression of matrilin-2 in oval cells during rat liver regeneration.

The matrilins represent a new family of oligomeric proteins that are assumed to act as adapter molecules connecting other proteins and proteoglycans in the extracellular matrix. Matrilin-2, the largest member of the family, displays a broad tissue distribution. It incorporates into loose and dense connective tissue and becomes associated with some basement membranes. The aim of our study was to analyse the expression of matrilin-2 in two liver regeneration models and to identify its cellular origin. Liver regeneration was induced in rats by partial hepatectomy (PH) and by the 2-acetylaminofluorene (AAF)/partial hepatectomy (PH) experimental models. Formalin fixed, paraffin embedded tissue sections were used for immunohistochemistry applying a rabbit matrilin-2 polyclonal antibody. Matrilin-2 was detected in normal rat liver and partially hepatectomized liver in the portal area, but could not be demonstrated in the acini. Matrilin-2 mRNA expression was analysed by RT-PCR and in situ hybridization. In the AAF/PH model the oval cells but not the hepatocytes produced matrilin-2 mRNA. Increase in protein level in the AAF/PH regenerating liver model was demonstrated by Western blotting. The protein was present in the basement membrane zone around the tubules formed by oval cells. Our data show that hepatic oval cells produce matrilin-2, a novel ECM protein, suggesting that matrilin-2 is an important component of ECM during stem cell-driven liver regeneration.

Imaging matrix metalloproteinase activity in multiple sclerosis as a specific marker of leukocyte penetration of the blood-brain barrier.

The enzymes gelatinase A/matrix metalloproteinase-2 (MMP-2) and gelatinase B/MMP-9 are essential for induction of neuroinflammatory symptoms in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS); in the absence of these enzymes, the disease does not develop. We therefore investigated the cellular sources and relative contributions of MMP-2 and MMP-9 to disease at early stages of EAE induction. We demonstrated that MMP-9 from an immune cell source is required in EAE for initial infiltration of leukocytes into the central nervous system and that MMP-9 activity is a reliable marker of leukocyte penetration of the blood-brain barrier. We then developed a molecular imaging method to visualize MMP activity in the brain using fluorescent- and radioactive-labeled MMP inhibitors (MMPis) in EAE animals and used the radioactive MMP ligand for positron emission tomography (PET) imaging of MMP activity in patients with MS. In contrast to traditional T1-gadolinium contrast-enhanced MRI, MMPi-PET enabled tracking of MMP activity as a unique feature of early lesions and ongoing leukocyte infiltration. MMPi-PET therefore allows monitoring of the early steps of MS development and provides a sensitive, noninvasive means of following lesion formation and resolution in murine EAE and human MS.