Basal membrane heparan sulphate proteoglycan expression during wound healing in human skin

Heparan sulphate proteoglycans (HSPGs) are integral components of the basement membrane (BM) in various tissues. HSPGs are important in the assembly and structure of the BM, and their putative functions include regulation of basement membrane permeability, binding of growth factors, and a role in cellular adhesion. In this study the expression of HSPG was examined during wound healing in human skin, using monoclonal antibodies (MAbs) that recognize the HSPG core protein and two different heparan sulphate (HS) epitopes, and the dynamics of HSPG expression were investigated in relation to epidermal cellular proliferation and permeability of the BM. Healing of excisional wounds in healthy volunteers was studied from day 0 up to 1 year. Intact human skin showed strong continuous staining of the dermo-epidermal BM and the vascular BM with all MAbs. Up to day 4 after wounding, staining for HSPG was absent under the ingrowing epidermis, with any of the MAbs, indicating that no complete BM was present. From day 7 onwards, the BM of the neo-epidermis showed positive staining for the HSPG core protein and a low sulphated HS epitope, and after day 14, the staining intensity was similar to normal skin. The staining patterns of these HSPG epitopes were similar to that of laminin. The staining pattern with a MAb against an epitope in the highly sulphated part of HS was found to be distinct from the other BM markers studied. This epitope was absent under the neo-epidermis up to 2 months after wounding. One year after wounding, the epitope was found to be present again. We observed that only in the time period between 2 months and 1 year had the epidermis normalized with respect to the number of cycling cells and the absence of high molecular weight plasma proteins. These findings suggest a correlation between normalization of epidermal proliferation, BM permeability, and regeneration of BM HS. It is proposed that complete BM maturation following skin wounding is a slow process and may account for the epidermal abnormalities that persist for a considerable period of time after wound healing. © 1997 John Wiley & Sons, Ltd.     

Prognostic implications of different cell cycle analysis models of flow cytometric DNA histograms of 1,301 breast cancer patients: Results from the multicenter morphometric mammary carcinoma project (MMMCP)

Conflicting prognostic results with regard to DNA flow cytometric cell cycle variables have been reported for breast cancer patients. An important reason for this may be related to differences in the interpretation of DNA histograms. Several computer programs based on different cell cycle fitting models are available resulting in significant variations in percent S-phase and other cell cycle variables. Our present study evaluated the prognostic value of percent S-phase cells obtained using 5 different cell cycle analysis models. Flow cytometric DNA histograms obtained from 1,301 fresh frozen breast cancer samples were interpreted with 5 different cell cycle analysis models using a commercially available computer program. Model 1 used the zero order S-phase calculation and “sliced nuclei” debris correction, model 2 added fixed G₂/M- to G₀/G₁-phase ratio, and model 3 added correction for aggregates. Model 4 applied the first-order S-phase calculation and sliced debris correction. Model 5 fixed the coefficients of variation CVs of the G₀/G₁- and G₂/M-phases in addition to applying the sliced nuclei debris correction and zero order S-phase calculation. The different models yielded clearly different prognostic results. The average percent S-phase cells of the aggregate correction model (model 3) provided the best prognostic value in all cases for overall survival (OS) as well as disease-free survival (DFS) (OS: p < 0.0001; DFS: p < 0.0001), in lymph node-positive cases (OS: p < 0.0001; DFS: p = 0.004) and in DNA-diploid subgroups (OS: p = 0.004; DFS: p = 0.001). For the lymph node negative and DNA-non-diploid subgroups, the percent S-phase of the second cell cycle reached slightly better prognostic significance than the average percent S-phase cells. In multivariate analysis, the average percent S-phase of the aggregate correction model had the best additional prognostic value to tumor size and lymph node status. In conclusion, different cell cycle analysis models yield clearly different prognostic results for invasive breast cancer patients. The most important prognostic percent S-phase variable was the average percent S-phase cells when aggregate correction was included in cell cycle analysis. Int. J. Cancer 74:260-269, 1997. © 1997 Wiley-Liss, Inc.     

Axonal damage in the spinal cord of MS patients occurs largely independent of T2 MRI lesions

OBJECTIVE: To determine the degree of axonal damage in relationship to signal abnormalities on T2-weighted high-resolution MRI in spinal cord tissue of patients with MS. METHODS: Spinal cord specimens of nine patients with MS and four controls were imaged at high resolution (4.7 T) in an axial plane and scored for lesions with increased signal intensity (SI). Histopathologic sections were cut and immunostained with NE14 (neurofilament marker) and Luxol fast blue (myelin stain). For each area, axonal density and diameter were quantified; axonal irregularity, NE14 axonal staining intensity, and myelin content were semiquantitatively scored. Included were 209 areas from MS cases and 109 areas from control cases distributed over lateral, posterior, and anterior columns. RESULTS: In control cases, no SI changes were found, average density of axons was 26,989/mm(2), average diameter was 1.1 micro m, and all scores for axonal irregularity, NE14 staining intensity, and myelin were normal. In MS cases, areas with increased SI were found, average axonal density was 11,807/mm(2) (p < 0.0001), and average axonal diameter 2.0 micro m (p = 0.001). Areas with high SI on MRI had lowest axonal density (average count: 10,504/mm(2); range: 3,433 to 26,325/mm(2)), largest diameter (average: 2.3 micro m; range: 1.0 to 4.0 micro m), and highest axonal irregularity and NE14 staining intensity compared to normal appearing cord tissue (NACT). However, NACT of MS cases also had lower axonal density (14,158/mm(2)) and higher average axonal diameter (1.6 micro m) than controls. CONCLUSIONS: Marked axonal loss occurs in MS spinal cords, largely independent of the degree of signal abnormality on T2-weighted MRI.     

Liposomes as drug carrier system for cis-diamminedichloroplatinum (II)

Summary In this study we have investigated the use of liposomes as a drug carrier system for cis-diamminedichloroplatinum (II) (cis-DDP) in order to reduce the nephrotoxicity with preservation of antitumor activity. Liposomes (PC/PS/Chol 10:1:4) were prepared using hydration media containing no or a relatively low concentration of NaCl. It was found that cis-DDP containing liposomes (lip cis-DDP) injected i.v. to IgM immunocytoma-bearing LOU/M rats at a dose of 1 mg cis-DDP/kg (cumulative dose 7 mg cis-DDP/kg) showed less antitumor activity than the free drug. The optimal cumulative dose of free cis-DDP for induction of antitumor activity in this tumor system is 7 mg/kg (7x1 mg/kg). At a dose of 2 mg lip cis-DDP/kg (cumulative dose 14 mg cis-DDP/kg) the antitumor activity was almost equal to that of free cis-DDP. The antitumor activity could not be increased by choosing another phospholipid composition of the liposomes [DPPC/DPPG/Chol (10:1:10)] cis-DPP incorporated in DPPC/ DPPG/Chol liposomes showed a similar antitumor activity to cis-DDP incorporated in PC/PS/Chol liposomes. After an i.v. dose of 2mg lip cis-DDP/kg (PC/PS/Chol) kidney damage was less compared to the treatment with free cis-DDP (1 mg/kg). However, after a single dose of 2 mg cis-DDP/kg or a cumulative dose of 8 or 16 mg cis-DDP/kg, kedneys of rats treated with lip cis-DDP contained twice as much Pt as after treatment with free cis-DDP. Moreover, after treatment with lip cis-DDP, a twofold increase of the amount of Pt in tumor tissue was measured. In vitro studies with Pt recovered from spleens obtained from rats treated with lip cis-DDP i.v. showed that based on the equal amounts of Pt recovered the antitumor activity of the recovered Pt was reduced, indicating inactivation of cis-DDP in vivo. As during treatment with free cis-DDP, recurrence of the tumor was observed during the continued treatment with lip cis-DDP. It was found that these recurrent tumors were resistant to further therapy with cis-DDP. In conclusion, cis-DDP encapsulation into liposomes decreased the nephrotoxicity. The antitumor activity of cis-DDP is preserved by liposome encapsulation when it was used at a dose of 2 mg/kg, but it was reduced in terms of earlier onset of regrowth.Abbreviations cis-DDP cis-Diamminedichloroplatinum(II) - Pt platinum - PC egg L--phosphatidylcholine - PS bovine L--phosphatidylserine - DPPC L--dipalmitoylphosphatidylcholine - DPPG L--dipalmitoylphosphatidylglycerol - Chol cholesterol - lip cis-DDP encapsulated in liposomes - PL phospholipid - AAS atomic absorption spectroscopy - RES reticuloendothelial system     

Progress and Future Prospectives in Skin-on-Chip Development with Emphasis on the use of Different Cell Types and Technical Challenges

Understanding the healthy and diseased state of skin is important in many areas of basic and applied research. Although the field of skin tissue engineering has advanced greatly over the last years, current in vitro skin models still do not mimic the complexity of the human skin. Skin-on-chip and induced pluripotent stem cells (iPSC) might be key technologies to improve in vitro skin models. This review summarizes the state of the art of in vitro skin models with regard to cell sources (primary, cell line, iPSC) and microfluidic devices. It can be concluded that iPSC have the potential to be differentiated into many kinds of immunologically matched cells and skin-on-chip technology might lead to more physiologically relevant skin models due to the controlled environment, possible exchange of immune cells, and an increased barrier function. Therefore the combination of iPSC and skin-on-chip is expected to lead to superior healthy and diseased in vitro skin models.     

The bone marrow constitutes a reservoir of pericyte progenitors

Adult bone marrow is a rich reservoir of hematopoietic and mesenchymal stem and progenitor cells. Mobilization and recruitment of bone marrow-derived cells to injured or ischemic tissue or tumors endorse the initiation and maintenance of angiogenic processes in the adult by incorporating endothelial progenitor cells (EPC) into the developing vasculature and by recruiting accessory hematopoietic cells. Recent data have now revealed that the origin of bone marrow-derived vascular cells is not restricted to endothelial cells but also includes pericytes--the perivascular support cells. Several laboratories have now reported the existence of pericyte progenitor cells, and these cells, like EPC, can be mobilized and recruited to the remodeling vasculature under ischemic conditions and in tumors. This review focuses on pericytes in vessel formation and on recent discoveries about their bone marrow origin in the adult.     

Development and application of monoclonal antibodies against SKALP/elafin and other trappin family members

SKALP/elafin is an epithelial proteinase inhibitor with antimicrobial properties that is not normally expressed in human epidermis, but is induced under inflammatory conditions and in some types of skin cancer. SKALP is a member of the recently described trappin gene family, which encodes a new class of proteins, characterized by a four-disulphide core and a transglutaminase substrate domain. Polyclonal antisera against SKALP have been shown to be useful for monitoring disease activity in psoriasis and tumour differentiation in squamous cell carcinoma. We developed ten different mouse monoclonal antibodies (mAbs) against synthetic peptides corresponding to a hexapeptide epitope in the transglutaminase substrate domain and three mAbs recognizing an epitope in the proteinase-inhibiting domain. The antibodies could be used with high specificity by immunohistochemistry on formalin-fixed tissue, by affinity chromatography, by Western blotting, and by enzyme-linked immunoadsorbent assay (ELISA) for the detection of SKALP/elafin. These antibodies have several advantages over existing polyclonal antisera, such as a defined epitope, the detection of full-length SKALP/elafin and unlimited supply. An antibody against the hexapeptide epitope, which is common to all known human, simian, bovine and swine trappin family members, was used to immunolocalize bovine trappins expressed in trachea, that have recently been discovered. These mAbs will serve as important new tools to measure SKALP/elafin and trappin family members in research and diagnostics.     

Reactivation of proliferin gene expression is associated with increased angiogenesis in a cell culture model of fibrosarcoma tumor progression

Proliferin (PLF) is an angiogenic placental hormone. We now report that PLF gene expression can also occur in a progressive fibrosarcoma mouse tumor cell model. PLF mRNA and protein are detectable at very low levels in cell lines derived from the mild noninvasive stage of tumor development. Expression is greatly augmented in cell lines from the aggressively invasive stage of development, a stage at which the tumor becomes highly angiogenic, and PLF expression remains high in cell lines from the end stage of fibrosarcoma. Activator protein 1 factors present at high levels in the more invasive stages of the tumor may in part allow for increased PLF expression, as cells from the mild stage in which c-jun and junB are stably expressed secrete levels of PLF comparable to that of the advanced stages. Secreted PLF protein is functionally important in tumor cell angiogenic activity, as demonstrated by the reduction of angiogenic activity in fibrosarcoma cell culture medium by immunodepletion of PLF. These results suggest that an extraembryonic genetic program, which has evolved to support fetal growth, may be reactivated in certain tumors and contribute to tumor growth.     

Vascular targeting of LIGHT normalizes blood vessels in primary brain cancer and induces intratumoural high endothelial venules

High‐grade brain cancer such as glioblastoma (GBM) remains an incurable disease. A common feature of GBM is the angiogenic vasculature, which can be targeted with selected peptides for payload delivery. We assessed the ability of micelle‐tagged, vascular homing peptides RGR, CGKRK and NGR to specifically bind to blood vessels in syngeneic orthotopic GBM models. By using the peptide CGKRK to deliver the tumour necrosis factor (TNF) superfamily member LIGHT (also known as TNF superfamily member 14; TNFSF14) to angiogenic tumour vessels, we have generated a reagent that normalizes the brain cancer vasculature by inducing pericyte contractility and re‐establishing endothelial barrier integrity. LIGHT‐mediated vascular remodelling also activates endothelia and induces intratumoural high endothelial venules (HEVs), which are specialized blood vessels for lymphocyte infiltration. Combining CGKRK–LIGHT with anti‐vascular endothelial growth factor and checkpoint blockade amplified HEV frequency and T‐cell accumulation in GBM, which is often sparsely infiltrated by immune effector cells, and reduced tumour burden. Furthermore, CGKRK and RGR peptides strongly bound to blood vessels in freshly resected human GBM, demonstrating shared peptide‐binding activities in mouse and human primary brain tumour vessels. Thus, peptide‐mediated LIGHT targeting is a highly translatable approach in primary brain cancer to reduce vascular leakiness and enhance immunotherapy. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.