Interstitial fluid pressure as an alternate regulator of angiogenesis independent of hypoxia driven HIF-1α in solid tumors.

We previously showed that interstitial fluid pressure (IFP) may be an alternate regulator of angiogenesis in solid tumors. Given the accepted link between hypoxia-induced factor and angiogenesis this study investigated the effect of IFP on hypoxia-inducible factor (HIF-1α) and vascular endothelial growth factor (VEGF) in human osteosarcoma xenografts in SCID mice and in different hypoxic environments. Tumors were grown either at heterotopic (flank) or orthotopic (medullary canal of the proximal tibia) sites in the host animal. Microfluidic probes determined pH, O(2)-saturation, IFP, and peripheral blood flow perfusion continuously. We assessed tumor growth in the orthotopic site (n = 15) by softex radiographs weekly, 3D microCT, histological evaluation, and for molecular responses. An increased cytoplasmic immunohistostaining of cells for HIF-1α (p = 0.03) and VEGF-A (p = 0.004) on the outer periphery was noted compared to the tumor center, with VEGFR2 uniformly stained throughout. This paralleled a raised state of interstitial hypertension (p = 0.007) in the tumor center relative to the peripheral surface but was inconsistent with a state of hypoxia (p = 0.03) in the tumor center. In vitro culture of human osteosarcoma cell lines (HOS, U2OS) and a human osteoblast control at 0- and 20-mmHg of hydrostatic pressure revealed suppression of HIF-1α (p = 0.02) and VEGF-A (p = 0.02) gene expression when IFP was raised, while the effect on VEGFR1 was equivocal. This study proposes an alternative regulatory angiogenic pathway via the influence of IFP on cancer cell function. The identification of a mechanistic cellular link to the physical parameter becomes an important tool to evaluate cancer cell growth within solid tumors.

Intramuscular nerve damage in lacerated skeletal muscles may direct the inflammatory cytokine response during recovery.

The expression of inflammatory cytokines and growth factors in surgically repaired lacerated muscles over a 12-week recovery phase was investigated. We hypothesized that these expression levels are influenced by both neural and muscular damage within lacerated muscles. Microarrays were confirmed with reverse transcription-polymerase chain reaction assays and histology of biopsies at the lesion of three simulated lacerated muscle models in 130 adult rats. The lacerated medial gastrocnemius with the main intramuscular nerve branch either cut (DN), crushed but leaving an intact nerve sheath (RN); or preserved intact (PN) were compared. At 4 weeks, DN had a higher number of interleukins up-regulated. DN and RN also had a set of Bmp genes significantly expressed between 2 and 8 weeks (P ≤ 0.05). By 12 weeks, DN had a poorer and slower myogenic recovery and greater fibrosis formation correlating with an up-regulation of the Tgf-ß gene family. DN also showed poorer re-innervation with higher mRNA expression levels of nerve growth factor (Ngf) and brain-derived neurotrophin growth factor (Bdnf) over RN and PN. This study demonstrates that the inflammatory response over 12 weeks in lacerated muscles may be directed by the type of intramuscular nerve damage, which can influence the recovery at the lesion site. Inflammatory-related genes associated to the type of intramuscular nerve damage include Gas-6, Artemin, Fgf10, Gdf8, Cntf, Lif, and Igf-2. qPCR also found up-regulation of Bdnf (1-week), neurotrophin-3 (2w), Lif (4w), and Ngf (4w, 8w) mRNA expressions in DN, making them possible candidates for therapeutic treatment to arrest the poor recovery in muscle lacerations (250).

Myosin heavy chain isoform profiles remain altered at 7 months if the lacerated medial gastrocnemius is poorly reinnervated: a study in rabbits.

Lacerated skeletal muscles often do not recover full function after repair. Denervated muscles with altered myosin heavy chain isoform (MHC) profiles are known to result in functional impairment. We studied the functional recovery of lacerated muscles, assessing MHC profile changes in association to the involvement of the intramuscular nerve (IM). We tested three lacerated models using the rabbit's medial gastrocnemius where the IM was either cut (NNR), repaired (NR), or preserved intact (NP). Muscles were assessed 7 months after repair for muscle atrophy, isometric contraction (by electrical stimulation), and fibrosis formation at the lesion site. Changes in myofibrillar actomyosin adenosine triphosphatase activity, MHC profile, regenerating myofibers and reinnervation were assessed by Western blot, histology, or immunohistology. Lacerated muscles with a repaired (NR) or an intact (NP) IM showed good recovery, with no significant changes in the MHC profile. Muscles where the IM was not repaired (NNR) resulted in significant scar area at the lesion site (p < 0.05), muscle atrophy (67%, p < 0.05) and loss in contractile properties (63% of the uninjured side, p < 0.05). At 7 months, all muscles were reinnervated. However, the NNR had an inappropriate (polyneural) and poorly distributed reinnervation, the presence of regenerating myofibers, and demonstrated a fast-to-slow MHC transition (71%:29% to 44%:56%, ANOVA, p = 0.018). This was associated to the cut IM when the NNR muscle was lacerated. Poor reinnervation in lacerated skeletal muscles alters the myosin heavy chain profile permanently. This study provides a rationale to also consider biological solutions to improve nerve regeneration and reinnervation in the surgical repair of lacerated muscles.

Upregulation of secretory connective tissue growth factor (CTGF) in keratinocyte-fibroblast coculture contributes to keloid pathogenesis.

Connective tissue growth factor (CTGF) plays a critical role in keloid pathogenesis by promoting collagen synthesis and deposition. Previous work suggested epithelial-mesenchymal interactions as a plausible factor affecting the expression of various growth factors and cytokines by both the epithelial and dermal mesenchymal cells. The aim of this study is to explore the role of epithelial-mesenchymal interactions in modulating CTGF expression. Immunohistochemistry was employed to check CTGF localization in skin tissue. Western blot assay was performed on total protein extracts from skin tissue, cell lysates and conditioned media to detect the basal/expression levels of CTGF. Study groups were subjected to serum stimulation (fibroblast-single cell culture) and pharmacological inhibitors targeted against mTOR (Rapamycin), Sp1 (WP631 and Mitoxanthrone), Smad3 (SB431542), and PI3K (LY294002). Increased localization of CTGF in the basal layer of keloid epidermis and higher expression of CTGF was observed in the keloid tissue extract. Interestingly, lower basal levels of CTGF was observed in fibroblast cell lysates cocultured with keloid keratinocytes compared to normal keratinocytes, while the conditioned media from the former culture consistently demonstrated a higher expression of secreted CTGF as compared to the latter group. These results demonstrate an important role of epithelial-mesenchymal interactions in the regulation of CTGF expression. Fibroblasts treated with inhibitors against mTOR, Sp1, Smad3, and PI3K demonstrated a reduced expression of CTGF, suggesting these signaling pathways to be important in the regulation of CTGF expression. Thus, revealing the therapeutic potentials for inhibitors that are selective for these factors in controlling CTGF expression in fibrotic conditions.

RUNX3, a novel tumor suppressor, is frequently inactivated in gastric cancer by protein mislocalization.

Loss of RUNX3 expression is suggested to be causally related to gastric cancer as 45% to 60% of gastric cancers do not express RUNX3 mainly due to hypermethylation of the RUNX3 promoter. Here, we examined for other defects in the properties of RUNX3 in gastric cancers that express RUNX3. Ninety-seven gastric cancer tumor specimens and 21 gastric cancer cell lines were examined by immunohistochemistry using novel anti-RUNX3 monoclonal antibodies. In normal gastric mucosa, RUNX3 was expressed most strongly in the nuclei of chief cells as well as in surface epithelial cells. In chief cells, a significant portion of the protein was also found in the cytoplasm. RUNX3 was not detectable in 43 of 97 (44%) cases of gastric cancers tested and a further 38% showed exclusive cytoplasmic localization, whereas only 18% showed nuclear localization. Evidence is presented suggesting that transforming growth factor-beta is an inducer of nuclear translocation of RUNX3, and RUNX3 in the cytoplasm of cancer cells is inactive as a tumor suppressor. RUNX3 was found to be inactive in 82% of gastric cancers through either gene silencing or protein mislocalization to the cytoplasm. In addition to the deregulation of mechanisms controlling gene expression, there would also seem to be at least one other mechanism controlling nuclear translocation of RUNX3 that is impaired frequently in gastric cancer.