Utility of Microvessel Density (MVD) by CD34 in Different Histological Grades of Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma is one of the leading causes of cancer related death and is preventable. Different grade at which a carcinoma presents determines the treatment modality and prognosis of the disease. Microvessel density measures formation of new vessels around tumor mass and is very important criteria for tumor metastasis and disease spread. Immunohistochemistry provide very definitive measures to determine the micro vessel density. Our study showed a positive correlation between Microvessel density and tumor size and dysplasia.

Efficacy of Cathelicidin LL-37 in an MRSA Wound Infection Mouse Model

Background: LL-37 is the only human antimicrobial peptide that belongs to the cathelicidins. The aim of the study was to evaluate the efficacy of LL-37 in the management of MRSA-infected surgical wounds in mice. Methods: A wound on the back of adult male BALB/c mice was made and inoculated with Staphylococcus aureus. Two control groups were formed (uninfected and not treated, C0; infected and not treated, C1) and six contaminated groups were treated, respectively, with: teicoplanin, LL-37, given topically and /or systemically. Histological examination of VEGF expression and micro-vessel density, and bacterial cultures of wound tissues, were performed. Results: Histological examination of wounds in the group treated with topical and intraperitoneal LL-37 showed increased re-epithelialization, formation of the granulation tissue, collagen organization, and angiogenesis. Conclusions: Based on the mode of action, LL-37 has a potential future role in the management of infected wounds.

Abstract 15873: Novel Contributions of Neutrophil-derived Myeloperoxidase and Hypochlorous Acid to 20-hydroxyeicosatetraenoic Acid Production That Drives Post-ischemic Angiogenesis

Introduction: Compensatory angiogenic response to ischemia is often insufficient in maintaining adequate tissue perfusion resulting in critical limb ischemia and amputation. Identifying a novel mechanism by which angiogenesis occurs in these conditions is clinically relevant. We recently uncovered that an increase in 20-HETE, an arachidonic acid metabolite of CYP4A/F ω-hydroxylases, regulates post-ischemic angiogenesis. However, the underlying mechanism resulting in this increase is unknown. Hypothesis: Neutrophil-derived myeloperoxidase (MPO) and hypochlorous acid (HOCl) critically contribute to post-ischemic 20-HETE increases that drive angiogenesis. Methods: Hindlimb ischemia was established in mice depleted of neutrophils, macrophages, and MPO (MPO -/- ). Angiogenesis was assessed by laser doppler perfusion imaging and micro-vessel density quantitation in the hindlimb gracilis muscles. MPO and HOCl were detected in these tissues using immunohistochemistry and a HOCl-specific fluorophore. We also determined the effects of MPO and HOCl on 20-HETE production, the expression of 20-HETE synthase CYP4A11, and hypoxia inducible factor-1α in cultured endothelial cells (EC) using LC/MS/MS, real time-PCR and western blot analysis, respectively. Results: We found that ischemia failed to increase 20-HETE production in mice depleted of neutrophils and MPO (13 ± 1.5 vs 35 ± 5 and ~2 ± .25 vs 35 ± 5 pg/mg of protein, respectively), accompanied with a decreased post-ischemic angiogenic phenotype. We also detected the formation of MPO and HOCl in post-ischemic gracilis muscles. MPO and HOCl also significantly stimulate CYP4A11 expression and 20-HETE production (40±12 vs 8±5 pg/mg of protein) in EC. Furthermore, HOCl quickly induces CYP4A11 mRNA/protein expression (2-fold,) and the protein expression of HIF-1α (2-fold) in as little as 15 min. Conclusion: Our studies establish for the first time that neutrophil-derived MPO and HOCl are responsible for promoting 20-HETE increases that critically drive angiogenesis post ischemia. Thus, identifying these novel mediators can further future therapeutic strategies to balance angiogenic responses during ischemia as well as treating diseases that are associated with abnormal angiogenesis.

Evaluation of angiogenesis as a prognostic marker in prostatic neoplasm especially carcinoma of prostate

Background: Prostatic carcinoma shows an unusually wide range of biological potential with well-known disparity between incidence and mortality for disease. Clinico-pathological studies suggests that angiogenesis and tumor neovascularity contributes to pathogenesis of prostate cancer. The aim of this study to present study was done to assess the validity of angiogenesis as a suitable prognostic marker in various prostatic disease specially the neoplasm’s including the malignant ones. Settings and design are Retrospective study.Methods: The present study of evaluation of angiogenesis as a prognostic marker in prostatic neoplasm especially carcinoma of prostate was done with 40 biopsy sample. The biopsy sample were obtained by suprapubic prostatectomy specimen and trans rectal needle biopsy specimen. Tissue sections were subjected to routine H and E staining. For demonstration of angiogenesis staining for reticulin fibers was applied.Results: The microvessels density increases as the severity of lesion increases from benign to pre-neoplastic to frankly malignant. The micro vessel density in malignant lesions is approximately thrice that in benign lesions. Conclusions: Very few studies have been done in prostatic lesions Hence an attempt is made to demonstrate and correlate angiogenesis as a tumour marker.

Enriched Populations of Human Megakaryocytic Cells Affect the Behavior of Myelofibrosis CD34+ Cells As Well As Cells Belonging to the MF Supportive Microenvironment

Objective The marrows and spleens of myelofibrosis (MF) patients are characterized by megakaryocytic (MK) hyperplasia as well as microenvironment (MicroE) abnormalities including increased micro-vessel density, stromal cell (SC) hyperplasia and fibrosis. MF is accompanied by dysregulation of inflammatory cytokines (INF-CyKs) which alter the tissue specific MicroE niches in which MF HSC reside. We hypothesized that MF MKs play a critical role in MF pathobiology and examined their potential to affect MF and normal CD34+ cells as well as marrow and splenic endothelial cells (EC) and SCs. Methods & Results MF MKP/MKs elaborate specific INF-CyKs. CD34+ cells were cultured in serum free medium with stem cell factor and thrombopoietin (TPO) for 7 days, and then cultured with TPO alone for an additional 7 days to generate cell population enriched for MK progenitor cells (MKPs) (CD34+CD41+) and MKs (CD34-/CD41+) (MKP/MK: 22-61% in normal and 27.6-54% in MF). MF MKP/MKs as compared to normal MKPs (nMKP/MKs) contained increased transcripts for several INF-CyKs including: IL-8 (31 fold), TGF-β (8 fold) and VEGF (93 fold). The transcripts for the P53 antagonist MDM2 (112 fold) and the activity of the transcription factor NF-κB were also increased. Furthermore, media conditioned by MF MKP/MKs contained increased protein levels of IL-8, TGF-β and VEGF (5.8, 1.4 and 5.2 fold, respectively) as compared to nMKP/MKs. Using immunohistochemistry, we demonstrated that IL-8 protein was present in normal and MF splenic SCs and ECs, but was exclusively present in MF splenic MKs. Plasma IL-8 levels were significantly elevated in MF patient plasma (p=0.0008). These data indicate that MF MKP/MKs elaborate a series of INF-CyKs which promote the development of MF. IL-8 promotes MF CD34+ cells proliferation. CXCR1 and CXCR2 are two receptors that bind IL-8. Flow cytometric analyses showed that MF CD34+ cells expressed higher levels of CXCR1 and CXCR2 than normal CD34+ cells (3.3 and 3.1 fold, respectively). Addition of IL-8 increased MF CD34+ cell numbers by 2 fold and assayable CFU-GM by 40% (p=0.033), but this effect was eliminated by the addition of reparixin, a CXCR1/CXCR2 antagonist (p=0.0055). MF MKP/MKs can alter the HSC MicroE. Using IHC and flow cytometry we observed that CXCR1 and CXCR2 were also expressed by splenic SCs and ECs and marrow mesenchymal stem cells. When SCs and ECs were incubated with equal numbers of normal or MF MKP/MKs, higher levels of SCF and VEGF were elaborated by SCs but not ECs. This effect was more pronounced with MF MKP/MKs as compared to nMKP/MKs (p=0.02 for SCF and p=0.003 for VEGF). By contrast, higher levels of IL-8 were elaborated by both ECs and SCs following co-cultivation with MF MKP/MKs (p=0.047 and p=0.03). The addition of reparixin to these co-cultures decreased the levels of VEGF and IL-8 to baseline. Co-culturing MF CD34+ cells with either SCs or ECs significantly increased the numbers of CD34+ cells, an effect which could be blocked by the addition of reparixin. These data indicate that MF MKP/MKs provide inflammatory signals that alter the MF MicroE which supports MF CD34+ cells and that these signals can be disrupted by drugs blocking IL-8-CXCR1/2 interactions. MF MKP/MKs and CD34+ cells can be targeted with ruxolitinib, the nutlin-RG7112 and a BET inhibitor. Treatment of MF CD34+ cells with low doses of RG7112, ruxolitinib, or JQ1 alone or in combinations decreased MF but not normal hematopoietic colony formation. Treatment of MF MKP/MKs with each of these agents decreased phosphor-NF-kB p65 levels as demonstrated by western blot and decreased the elaboration of IL-8 by 20 to 60%. Conclusion These data indicate that MF MKPs are characterized by increased transcripts for MDM2 as well as NF-κB activity contributing / leading to the MK hyperplasia characteristic of MF and the elaboration of a group of lineage specific cytokines (TGF-β, IL-8, VEGF) that not only affect MF CD34+ cells but also cells belonging to the MicroE (ECs and SCs) (Figure 1). The MF promoting activities of MF MKP/MKs can be effectively targeted with ruxolitinib, RG7112, and BET inhibitors. Furthermore reparixin can be utilized to interrupt the interactions between IL-8 and CXCR1/2 expressing cells (CD34+ cells, ECs and SCs). These data provide the preclinical rationale for the evaluation of combinations of drugs which can dampen the cascade of events that result when MF MKP/MKs interact with CD34+ cells and MicroE cells. Disclosures Hoffman: Summer Road: Research Funding; Merus: Research Funding; Janssen: Research Funding; Incyte: Research Funding; Formation Biologics: Research Funding.