A Combination therapy using an mTOR inhibitor and Honokiol effectively induces autophagy through the modulation of AXL and Rubicon in renal cancer cells and restricts renal tumor growth following organ transplantation.

Development of cancer, including renal cancer, is a major problem in immunosuppressed patients. The mTOR inhibitor Rapamycin (RAPA) is used as an immunosuppressive agent in patients with organ transplants and other immunological disorders; and it also has antitumorigenic potential. However, long-term use of RAPA causes reactivation of Akt, and ultimately leads to enhanced tumor growth. Honokiol (HNK) is a natural compound, which possesses both anti-inflammatory and antitumorigenic properties. In this study, we investigated the effect of a novel combination therapy using RAPA + HNK on allograft survival and post-transplantation renal tumor growth. We observed that it effectively modulated the expression of some key regulatory molecules (like Carabin, an endogenous Ras inhibitor; and Rubicon, a negative regulator of autophagy) that play important roles in tumor cell growth and survival. This combination induced toxic autophagy and apoptosis to promote cancer cell death; and was associated with a reduced expression of the tumor-promoting receptor tyrosine kinase AXL. Finally, we utilized a novel murine model to examine the effect of RAPA + HNK on post-transplantation renal tumor growth. The combination treatment prolonged the allograft survival and significantly inhibited post-transplantation tumor growth. It was associated with reduced tumor expression of Rubicon and the cytoprotective/antioxidant heme oxygenase-1 to overcome therapeutic resistance. It also downregulated the coinhibitory programmed death-1 ligand, which plays major role(s) in the immune escape of tumor cells. Together, this combination treatment has a great potential to restrict renal tumor growth in transplant recipients as well as other immunosuppressed patients.

Intraarticular injection of relaxin-2 alleviates shoulder arthrofibrosis.

Arthrofibrosis is a prevalent condition affecting greater than 5% of the general population and leads to a painful decrease in joint range of motion (ROM) and loss of independence due to pathologic accumulation of periarticular scar tissue. Current treatment options are limited in effectiveness and do not address the underlying cause of the condition: accumulation of fibrotic collagenous tissue. Herein, the naturally occurring peptide hormone relaxin-2 is administered for the treatment of adhesive capsulitis (frozen shoulder) and to restore glenohumeral ROM in shoulder arthrofibrosis. Recombinant human relaxin-2 down-regulates type I collagen and α smooth muscle actin production and increases intracellular cAMP concentration in human fibroblast-like synoviocytes, consistent with a mechanism of extracellular matrix degradation and remodeling. Pharmacokinetic profiling of a bolus administration into the glenohumeral joint space reveals the brief systemic and intraarticular (IA) half-lives of relaxin-2: 0.96 h and 0.62 h, respectively. Furthermore, using an established, immobilization murine model of shoulder arthrofibrosis, multiple IA injections of human relaxin-2 significantly improve ROM, returning it to baseline measurements collected before limb immobilization. This is in contrast to single IA (sIA) or multiple i.v. (mIV) injections of relaxin-2 with which the ROM remains constrained. The histological hallmarks of contracture (e.g., fibrotic adhesions and reduced joint space) are absent in the animals treated with multiple IA injections of relaxin-2 compared with the untreated control and the sIA- and mIV-treated animals. As these findings show, local delivery of relaxin-2 is an innovative treatment of shoulder arthrofibrosis.

T Cell-Specific Adaptor Protein Regulates Mitochondrial Function and CD4+ T Regulatory Cell Activity In Vivo following Transplantation.

The T cell-specific adaptor protein (TSAd), encoded by the SH2D2A gene, is an intracellular molecule that binds Lck to elicit signals that result in cytokine production in CD4+ T effector cells (Teff). Nevertheless, using Sh2d2a knockout (KO; also called TSAd-/-) mice, we find that alloimmune CD4+ Teff responses are fully competent in vivo. Furthermore, and contrary to expectations, we find that allograft rejection is accelerated in KO recipients of MHC class II-mismatched B6.C-H-2bm12 heart transplants versus wild-type (WT) recipients. Also, KO recipients of fully MHC-mismatched cardiac allografts are resistant to the graft-prolonging effects of costimulatory blockade. Using adoptive transfer models, we find that KO T regulatory cells (Tregs) are less efficient in suppressing Teff function and they produce IFN-γ following mitogenic activation. In addition, pyrosequencing demonstrated higher levels of methylation of CpG regions within the Treg-specific demethylated region of KO versus WT Tregs, suggesting that TSAd, in part, promotes Treg stability. By Western blot, Lck is absent in the mitochondria of KO Tregs, and reactive oxygen species production by mitochondria is reduced in KO versus WT Tregs. Full transcriptomic analysis demonstrated that the key mechanism of TSAd function in Tregs relates to its effects on cellular activation rather than intrinsic effects on mitochondria/metabolism. Nevertheless, KO Tregs compensate for a lack of activation by increasing the number of mitochondria per cell. Thus, TSAd serves as a critical cell-intrinsic molecule in CD4+Foxp3+ Tregs to regulate the translocation of Lck to mitochondria, cellular activation responses, and the development of immunoregulation following solid organ transplantation.

DEPTOR regulates vascular endothelial cell activation and proinflammatory and angiogenic responses.

The maintenance of normal tissue homeostasis and the prevention of chronic inflammatory disease are dependent on the active process of inflammation resolution. In endothelial cells (ECs), proinflammation results from the activation of intracellular signaling responses and/or the inhibition of endogenous regulatory/pro-resolution signaling networks that, to date, are poorly defined. In this study, we find that DEP domain containing mTOR interacting protein (DEPTOR) is expressed in different microvascular ECs in vitro and in vivo, and using a small interfering RNA (siRNA) knockdown approach, we find that it regulates mammalian target of rapamycin complex 1 (mTORC1), extracellular signal-regulated kinase 1/2, and signal transducer and activator of transcription 1 activation in part through independent mechanisms. Moreover, using limited gene arrays, we observed that DEPTOR regulates EC activation including mRNA expression of the T-cell chemoattractant chemokines CXCL9, CXCL10, CXCL11, CX3CL1, CCL5, and CCL20 and the adhesion molecules intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 (P < .05). DEPTOR siRNA-transfected ECs also bound increased numbers of peripheral blood mononuclear cells (P < .005) and CD3+ T cells (P < .005) in adhesion assays in vitro and had increased migration and angiogenic responses in spheroid sprouting (P < .01) and wound healing (P < .01) assays. Collectively, these findings define DEPTOR as a critical upstream regulator of EC activation responses and suggest that it plays an important role in endogenous mechanisms of anti-inflammation and pro-resolution.

The cn/cn dwarf mouse. Histomorphometric, ultrastructural, and radiographic study in mutants corresponding to human acromesomelic dysplasia Maroteaux type (AMDM).

BACKGROUND: The cn/cn dwarf mouse is caused by a loss-of-function mutation in the natriuretic peptide receptor 2 (NPR-2) gene which helps positively regulate endochondral longitudinal bone growth. The gene mutation corresponds to that in the human skeletal dysplasia Acromesomelic Dysplasia Maroteaux type (AMDM). This study assesses histomorphometric, ultrastructural and radiographic correlates of the growth abnormality. METHODS: Ten litters of cn/cn and cn/+littermates at ages ranging from 2.5 to 6.5 weeks were studied by skeletal radiographs, histomorphometry and physeal ultrastructure. Skeletal radiographs were done on 2 cn/cn and 2 cn/+littermates at 5 weeks of age. Humeral, femoral, and tibial lengths were measured from 34 intact bones (17 cn/cn, 17 cn/+) at 2.5 to 6.5 weeks. Growth plate histomorphometry in 50 bones (26 cn/cn and 24 cn/+) determined the hypertrophic zone/entire physeal cartilage ratios in 204 sections (87 cn/+, 117 cn/cn) at 3 time periods (2.5-3, 4-4.5, and 6-6.5 weeks). Electron microscopy assessed 6 cn/cn and 6 cn/+age and site-matched physeal cartilage. RESULTS: Cn/cn mice were two thirds the size of the cn/+. Cn/cn bones were normal in shape or only minimally deformed except for the radius with mid-diaphyseal bowing. Length ratios of cn/cn humeri, femurs, and tibias were a mean of 0.65 (± 0.03, n = 34, 17 ratios) compared to cn/+bones. The main physeal abnormality was a markedly shortened hypertrophic zone with the ratio of hypertrophic zone to entire physis 0.17 (± 0.063) in the cn/cn and 0.30 (± 0.052) in the cn/+mice. Ratio assessments were similar comparing humeral, femoral, and tibial growth plates as were ratios from each of the 3 time periods. Ultrastructural assessments from the resting zone to the lower hypertrophic zone-metaphyseal junction showed no specific individual cell abnormalities in cn/cn compared to cn/+physes. CONCLUSIONS: The disorder causes a shortened physeal hypertrophic zone but normal ultrastructure of cn/cn chondrocytes points to abnormality primarily affecting the hypertrophic zone rather than a structural cell or matrix synthesis problem.

Pudgy mouse rib deformities emanate from abnormal paravertebral longitudinal cartilage/bone accumulations.

The pudgy (pu/pu) mouse, caused by a recessive mutation in the Notch family Delta like-3 gene (Dll3), has severe rib, vertebral body and intervertebral disc abnormalities. Using whole-mount preparations and serial histologic sections we demonstrate: 1) localized paravertebral longitudinal cartilage/bone accumulations (PVLC/BAs) invariably associated with branched, fused and asymmetrically spaced ribs that emanate from it laterally; 2) abnormal rib formation immediately adjacent to abnormal vertebral body and intervertebral disc formation in asymmetric right/left fashion; and 3) patterns of rib deformation that differ in each mouse. Normal BALB/c embryo and age-matched non-affected pu/+ mice assessments allow for pu/pu comparisons. The Dll3 Notch family gene is involved in normal somitogenesis via the segmentation clock mechanism. Although pathogenesis of rib deformation is initially triggered by the Dll3 gene mutation, these findings of abnormal asymmetric costo-vertebral region structure imply that differing patterns cannot be attributed to this single gene mutation alone. All findings implicate a dual mechanism of malformation: the Dll3 gene mutation leading to subtle timing differences in traveling oscillation waves of the segmentation clock and further subsequent misdirection of tissue formation by altered chemical reaction-diffusion and epigenetic landscape responses. PVLC/BAs appear as primary supramolecular structures underlying severe rib malformation associated both with time-sensitive segmentation clock mutations and subsequent reactions.

A peptide trivalent arsenical inhibits tumor angiogenesis by perturbing mitochondrial function in angiogenic endothelial cells.

Mitochondria are the powerhouse of the cell and their disruption leads to cell death. We have used a peptide trivalent arsenical, 4-(N-(S-glutathionylacetyl)amino) phenylarsenoxide (GSAO), to inactivate the adenine nucleotide translocator (ANT) that exchanges matrix ATP for cytosolic ADP across the inner mitochondrial membrane and is the key component of the mitochondrial permeability transition pore (MPTP). GSAO triggered Ca(2+)-dependent MPTP opening by crosslinking Cys(160) and Cys(257) of ANT. GSAO treatment caused a concentration-dependent increase in superoxide levels, ATP depletion, mitochondrial depolarization, and apoptosis in proliferating, but not growth-quiescent, endothelial cells. Endothelial cell proliferation drives new blood vessel formation, or angiogenesis. GSAO inhibited angiogenesis in the chick chorioallantoic membrane and in solid tumors in mice. Consequently, GSAO inhibited tumor growth in mice with no apparent toxicity at efficacious doses.

Histopathology of osteogenesis imperfecta bone. Supramolecular assessment of cells and matrices in the context of woven and lamellar bone formation using light, polarization and ultrastructural microscopy.

Diaphyseal long bone cortical tissue from 30 patients with lethal perinatal Sillence II and progressively deforming Sillence III osteogenesis imperfecta (OI) has been studied at multiple levels of structural resolution. Interpretation in the context of woven to lamellar bone formation by mesenchymal osteoblasts (MOBLs) and surface osteoblasts (SOBLs) respectively demonstrates lamellar on woven bone synthesis as an obligate self-assembly mechanism and bone synthesis following the normal developmental pattern but showing variable delay in maturation caused by structurally abnormal or insufficient amounts of collagen matrix. The more severe the variant of OI is, the greater the persistence of woven bone and the more immature the structural pattern; the pattern shifts to a structurally stronger lamellar arrangement once a threshold accumulation for an adequate scaffold of woven bone has been reached. Woven bone alone characterizes lethal perinatal variants; variable amounts of woven and lamellar bone occur in progressively deforming variants; and lamellar bone increasingly forms rudimentary and then partially compacted osteons not reaching full compaction. At differing levels of microscopic resolution: lamellar bone is characterized by short, obliquely oriented lamellae with a mosaic appearance in progressively deforming forms; polarization defines tissue conformations and localizes initiation of lamellar formation; ultrastructure of bone forming cells shows markedly dilated rough endoplasmic reticulum (RER) and prominent Golgi bodies with disorganized cisternae and swollen dispersed tubules and vesicles, structural indications of storage disorder/stress responses and mitochondrial swelling in cells with massively dilated RER indicating apoptosis; ultrastructural matrix assessments in woven bone show randomly oriented individual fibrils but also short pericellular bundles of parallel oriented fibrils positioned obliquely and oriented randomly to one another and in lamellar bone show unidirectional fibrils that deviate at slight angles to adjacent bundles and obliquely oriented fibril groups consistent with twisted plywood fibril organization. Histomorphometric indices, designed specifically to document woven and lamellar conformations in normal and OI bone, establish ratios for: i) cell area/total area X 100 indicating the percentage of an area occupied by cells (cellularity index) and ii) total area/number of cells (pericellular matrix domains). Woven bone is more cellular than lamellar bone and OI bone is more cellular than normal bone, but these findings occur in a highly specific fashion with values (high to low) encompassing OI woven, normal woven, OI lamellar and normal lamellar conformations. Conversely, for the total area/number of cells ratio, pericellular matrix accumulations in OI woven are smallest and normal lamellar largest. Since genotype-phenotype correlation is not definitive, interposing histologic/structural analysis allowing for a genotype-histopathologic-phenotype correlation will greatly enhance understanding and clinical management of OI.

CD40-induced signaling in human endothelial cells results in mTORC2- and Akt-dependent expression of vascular endothelial growth factor in vitro and in vivo.

We have examined CD40-dependent signals in endothelial cells (EC) mediating the expression of vascular endothelial growth factor (VEGF) and VEGF-induced angiogenesis. We treated confluent cultures of EC with soluble CD40L (sCD40L), and by Western blot found a marked increase in the phosphorylation of Akt, 4EBP-1, and S6K1, compared with untreated cells. EC were transfected with a full-length VEGF promoter-luciferase construct and cultured in the absence or presence of rapamycin and sCD40L. We found that rapamycin, which blocks mTORC1 and mTORC2 signaling, inhibited sCD40L-mediated transactivation of VEGF. In addition, by Western blot, we found that the transfection of EC with small interfering RNA (siRNA) to rictor (to inhibit mTORC2), and not raptor (to inhibit mTORC1), inhibited sCD40L-dependent protein expression of VEGF. In additions, we found that basal levels of phosphorylated Akt as well as VEGF were increased in EC transfected with the raptor siRNA. Also, rapamycin failed to inhibit VEGF promoter activation, as well as VEGF protein expression in EC transfected with a constitutively active construct of Akt, further demonstrating that mTORC1 is not necessary for CD40- and Akt-induced expression of VEGF. Finally, we injected human CD40L-transfected fibroblasts or mock transfectants into human skin on SCID mice. We found that the injection of CD40L transfectants, but not mock cells, resulted in VEGF expression and mediated a marked angiogenesis reaction, and this response was reduced in mice treated with rapamycin. Together, these observations indicate that mTORC2 and Akt facilitate CD40-inducible expression of VEGF in EC, which is of clinical importance in tumor growth and the progression of chronic inflammatory diseases.

Molecular differentiation in epiphyseal and physeal cartilage. Prominent role for gremlin in maintaining hypertrophic chondrocytes in epiphyseal cartilage.

We have studied hypertrophic and immediately adjacent pre-hypertrophic chondrocytes at the same stage of histologic development in 7 day old post-natal Balb/C mouse physes and epiphyses. Laser capture microdissection (LCM) and GeneChip microarray analysis compared the molecular composition of the two hypertrophic chondrocyte regions. Molecules upregulated in dramatically higher levels in the epiphysis were gremlin (58-fold), epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (25-fold), and frizzled related protein (6.4-fold and 5.7-fold). Molecules upregulated in higher levels in the physis were proline arginine-rich end leucine-rich repeat protein (PRELP) (15.6-fold), pyrophosphatase (inorganic) 1 (10-fold) and hedgehog-interacting protein (7.3-fold). Immunocytochemistry for gremlin confirmed specific localization patterns. This study indicates a critical site-specific role for hypertrophic chondrocytes with different synthesis patterns in separate regions even though they appear structurally the same and are at the same stage of development.

Activation of c-Met in cancer cells mediates growth-promoting signals against oxidative stress through Nrf2-HO-1.

Any imbalance between reactive oxygen species (ROS) generation and the anti-oxidant capacity lead to cellular oxidative stress. Many chemotherapeutic agents mediate their cytotoxic functions through the generation of ROS. c-Met, a receptor tyrosine kinase, is over-expressed in renal cancer and plays very crucial role(s) in its growth and survival. Here, we show that c-Met activation protected renal cancer cells from ROS, oxidative stress and cytotoxicity induced by the anti-cancer agent sorafenib (used for renal cancer treatment); and it markedly attenuated sorafenib-induced DNA damage. Activated c-Met promoted the anti-apoptotic proteins (Bcl-2 and Bcl-xL) and inhibited apoptotic cleaved caspase-3. We found that the cytoprotective function of c-Met against sorafenib-induced ROS generation and apoptosis was mediated primarily through the activation of anti-oxidant Nrf2-HO-1. c-Met promoted the nuclear localization of Nrf2 and hindered its binding with the inhibitory protein Keap1. Silencing of Nrf2 attenuated the protective action of c-Met against sorafenib-induced oxidative stress. To evaluate the physiological significance of our findings, in a tumor xenograft model, we observed that a combination treatment with pharmacological inhibitors of c-Met and it's anti-oxidant downstream effecter HO-1 markedly reduced the growth of renal tumor in vivo; it increased the oxidative stress, DNA damage and apoptotic markers in the tumor xenografts, along with reduced tumor vessel density. Our observations indicate that the c-Met-Nrf2-HO-1 pathway plays a vital role in relieving ROS-mediated oxidative stress of renal tumors. Targeting this pathway can significantly increase the oxidative stress to promote apoptotic death of cancer cells.

Inhibition of intra-abdominal adhesion formation with the angiogenesis inhibitor sunitinib.

OBJECTIVE: To determine the effects of sunitinib, a vascular endothelial growth factor receptor 2 (VEGFR-2) antagonist, on intra-abdominal adhesions. BACKGROUND: In the United States, complications from adhesions cost $1 billion and account for 846,000 inpatient days annually. Endothelial mitogens, such as VEGF, are up-regulated during adhesion formation. Sunitinib, a tyrosine kinase inhibitor with antiangiogenic and antitumor properties, may prevent or reduce postoperative abdominal adhesions by VEGFR-2 inhibition. METHODS: The cecum of 37 mice were abraded to promote adhesion formation and a silicone patch was sutured to the abdominal wall. The mice were randomized into two groups: Group 1 was treated with sunitinib in methylcellulose by oral gavage daily and Group 2 (control) received methylcellulose alone. After 10 d the mice were sacrificed and intra-abdominal adhesions were scored. The experiment was then repeated and mice were sacrificed on postoperative day 30 to assess the long-term effects of sunitinib. RESULTS: All 19 control mice developed intra-abdominal adhesions. Six of the 18 (33.3%) mice in the treatment group were adhesion-free. Collectively, the sunitinib-treated mice had a lower adhesion score [2.0 (IQR 0.0-5.0; range 0-8.0)] than the control group [5.0 (IQR 3.0-8.0; range 2.0-10.0) (P = 0.002)]. Long-term results were consistent with this finding [sunitinib 0.0 (IQR 0.0-3.0; range 0-7) and control 6.0 (IQR 3.0-7.0; range 0-12) (P = 0.049)]. CONCLUSION: Adhesion formation is angiogenesis-dependent and is in part mediated through VEGFR-2. Sunitinib, a VEGFR-2 antagonist, significantly reduces adhesion formation in a murine model. Antiangiogenic therapy may be an efficacious strategy to prevent or treat adhesions after intra-abdominal procedures.

Organ-specific differences in the function of MCP-1 and CXCR3 during cardiac and skin allograft rejection.

BACKGROUND: Chemokines are well-established to function in the recruitment of leukocytes into allografts in the course of rejection. Moreover, some studies have indicated that there are organ-specific differences in chemokine function, but the mechanism accounting for this difference is not known. METHODS: Fully major histocompatibility complex-mismatched vascularized cardiac transplants or skin transplants were performed using BALB/c (H-2d), C57BL/6 (H-2b), MCP-1-/- (H-2b) and CXCR3-/- (H-2b) mice as donors or recipients. Also, skin grafts (H-2b) were placed onto SCID mice (H-2d) that received BALB/c splenocytes (H-2d) by adoptive transfer either at the time of transplantation, or after a period of 28 days. RESULTS: Cardiac allografts in MCP-1-/- recipients survived significantly longer (P<0.0005) than wild-type (WT) controls. However, there was no prolongation of survival when MCP-1-/- grafts were used a donors in WT mice. In contrast, the absence of donor but not recipient MCP-1 prolonged skin allograft survival. WT donor cardiac grafts in CXCR3-/- recipients had a modest prolongation of survival (P<0.0005), whereas CXCR3-/- donor cardiac grafts in WT recipients were rejected similar to controls. Also, while recipient CXCR3 had no effect on the rejection of skin, CXCR3-/- donor skin grafts survived significantly longer than WT controls. This survival advantage was lost when vascularized CXCR3-/- skin grafts were used as donors in the SCID model of rejection. CONCLUSION: Recipient derived MCP-1 and CXCR3 are functional in the rejection of vascularized, but not nonvascularized, allografts. In contrast, donor-derived MCP-1 and CXCR3 are functional in nonvascularized, but not vascularized grafts.

Honokiol inhibits c-Met-HO-1 tumor-promoting pathway and its cross-talk with calcineurin inhibitor-mediated renal cancer growth.

Honokiol (HNK) is a small molecule with potent anti-inflammatory and anti-tumorigenic properties; yet the molecular targets of HNK are not well studied. Hyperactivation of the receptor tyrosine kinase c-Met and overexpression of the cytoprotective enzyme heme oxygenase-1 (HO-1) play a critical role in the growth and progression of renal cell carcinoma (RCC). Interestingly, the calcineurin inhibitor (CNI) cyclosporine A (CsA), an immunosuppressant used to prevent allograft rejection, can also increase the risk of RCC in transplant patients. We studied the potential role of c-Met signaling axis on CNI-induced renal tumor growth and tested the anti-tumor efficacy of HNK. Importantly, CNI treatment promoted c-Met induction and enhanced c-Met-induced Ras activation. We found that HNK treatment effectively down-regulated both c-Met phosphorylation and Ras activation in renal cancer cells. It inhibited the expression of both c-Met- and CNI-induced HO-1, and promoted cancer cell apoptosis. In vivo, HNK markedly inhibited CNI-induced renal tumor growth; and it decreased the expression of phospho-c-Met and HO-1 and reduced blood vessel density in tumor tissues. Our results suggest a novel mechanism(s) by which HNK exerts its anti-tumor activity through the inhibition of c-Met-Ras-HO-1 axis; and it can have significant therapeutic potential to prevent post-transplantation cancer in immunosuppressed patients.

Structural differences in epiphyseal and physeal hypertrophic chondrocytes.

We have observed that epiphyseal and physeal hypertrophic chondrocytes in BALB/c mice show considerable differences of light microscopic and ultrastructural appearance, even when the cells are at the same stage of differentiation. In addition, cell structure maintenance improved with tissue preparation controlled for osmolarity and for membrane stabilization using 0.5% ruthenium hexammine trichloride (RHT) for both light microscopy (LM) and electron microscopy (EM) or 0.5% lanthanum nitrate for LM. Physeal hypertrophic chondrocytes showed a gradual increase in size closer to the metaphysis and a change in shape as cells elongated along the long axis. The nucleus remained central, with uniformly dispersed chromatin, and the rough endoplasmic reticulum (RER) was randomly dispersed throughout cytoplasm with little to no presence against the cell membrane. Even the lowermost cells showed thin elongated or dilated cisternae of RER and intact cell membranes. Epiphyseal chondrocytes remained circular to oval with no elongation. Nucleus and RER were positioned as a complete transcellular central nucleocytoplasmic column or as an incomplete bud with RER of the column/bud always continuous with RER peripherally against the intact cell membrane. RER was densely packed with parallel cisternae with adjacent cytoplasm empty of organelles but often filled with circular deposits of moderately electron-dense material consistent with fat. Optimal technique for LM involved fixation using glutaraldehyde (GA) 1.3%, paraformaldehyde (PFA) 1% and RHT 0.5% (mOsm 606) embedded in JB-4 plastic and stained with 0.5% toluidine blue. Optimal technique for EM used fixation with GA 1.3%, PFA 1%, RHT 0.5% and cacodylate buffer 0.03 M (mOsm 511) and post-fixation including 1% osmium tetroxide. These observations lead to the possibility that the same basic cell, the hypertrophic chondrocyte, has differing functional mechanisms at different regions of the developing bone.

Vitamin D binding protein-macrophage activating factor (DBP-maf) inhibits angiogenesis and tumor growth in mice.

We have isolated a selectively deglycosylated form of vitamin D binding protein (DBP-maf) generated from systemically available DBP by a human pancreatic cancer cell line. DBP-maf is antiproliferative for endothelial cells and antiangiogenic in the chorioallantoic membrane assay. DBP-maf administered daily was able to potently inhibit the growth of human pancreatic cancer in immune compromised mice (T/C=0.09). At higher doses, DBP-maf caused tumor regression. Histological examination revealed that treated tumors had a higher number of infiltrating macrophages as well as reduced microvessel density, and increased levels of apoptosis relative to untreated tumors. Taken together, these data suggest that DBP-maf is an antiangiogenic molecule that can act directly on endothelium as well as stimulate macrophages to attack both the endothelial and tumor cell compartment of a growing malignancy.

Effectiveness of a combination therapy using calcineurin inhibitor and mTOR inhibitor in preventing allograft rejection and post-transplantation renal cancer progression.

Calcineurin inhibitors (CNIs) may promote post-transplantation cancer through altered expression of cytokines and chemokines in tumor cells. We found that there is a potential cross-talk among CNI-induced signaling molecules and mTOR. Here, we utilized a murine model of post-transplantation cancer to examine the effect of a combination therapy (CNI + mTOR-inhibitor rapamycin) on allograft survival and renal cancer progression. The therapy prolonged allograft survival; and significantly attenuated CNI-induced post-transplantation cancer progression, with down-regulation of mTOR and S6-kinase phosphorylation. Also, rapamycin inhibited CNI-induced over-expression of the angiogenic cytokine VEGF, and the chemokine receptor CXCR3 and its ligands in post-transplantation tumor tissues.

Calcineurin inhibitor-induced and Ras-mediated overexpression of VEGF in renal cancer cells involves mTOR through the regulation of PRAS40.

Malignancy is a major problem in patients treated with immunosuppressive agents. We have demonstrated that treatment with calcineurin inhibitors (CNIs) can induce the activation of proto-oncogenic Ras, and may promote a rapid progression of human renal cancer through the overexpression of vascular endothelial growth factor (VEGF). Interestingly, we found that CNI-induced VEGF overexpression and cancer cell proliferation was inhibited by rapamycin treatment, indicating potential involvement of the mammalian target of rapamycin (mTOR) pathway in this tumorigenic process. Here, we examined the role of mTOR pathway in mediating CNI- and Ras-induced overexpression of VEGF in human renal cancer cells (786-0 and Caki-1). We found that the knockdown of raptor (using siRNA) significantly decreased CNI-induced VEGF promoter activity as observed by promoter-luciferase assay, suggesting the role of mTOR complex1 (mTORC1) in CNI-induced VEGF transcription. It is known that mTOR becomes activated following phosphorylation of its negative regulator PRAS40, which is a part of mTORC1. We observed that CNI treatment and activation of H-Ras (through transfection of an active H-Ras plasmid) markedly increased the phosphorylation of PRAS40, and the transfection of cells using a dominant-negative plasmid of Ras, significantly decreased PRAS40 phosphorylation. Protein kinase C (PKC)-ζ and PKC-δ, which are critical intermediary signaling molecules for CNI-induced tumorigenic pathway, formed complex with PRAS40; and we found that the CNI treatment increased the complex formation between PRAS40 and PKC, particularly (PKC)-ζ. Inhibition of PKC activity using pharmacological inhibitor markedly decreased H-Ras-induced phosphorylation of PRAS40. The overexpression of PRAS40 in renal cancer cells significantly down-regulated CNI- and H-Ras-induced VEGF transcriptional activation. Finally, it was observed that CNI treatment increased the expression of phosho-PRAS40 in renal tumor tissues in vivo. Together, the phosphorylation of PRAS40 is critical for the activation of mTOR in CNI-induced VEGF overexpression and renal cancer progression.

Femoral head deformation and repair following induction of ischemic necrosis: a histologic and magnetic resonance imaging study in the piglet.

BACKGROUND: Ischemic necrosis of the femoral head can be induced surgically in the piglet. We used this model to assess femoral head deformation and repair in vivo by sequential magnetic resonance imaging and by correlating end-stage findings with histologic assessments. METHODS: Ischemic necrosis of the femoral head was induced in ten three-week-old piglets by tying a silk ligature around the base of the femoral neck (intracapsular) and cutting the ligamentum teres. We used magnetic resonance imaging with the piglets under general anesthesia to study the hips at forty-eight hours and at one, two, four, and eight weeks. Measurements on magnetic resonance images in the midcoronal plane of the involved and control sides at each time documented the femoral head height, femoral head width, superior surface cartilage height, and femoral neck-shaft angle. Histologic assessments were done at the time of killing. RESULTS: Complete ischemia of the femoral head was identified in all involved femora by magnetic resonance imaging at forty-eight hours. Revascularization began at the periphery of the femoral head as early as one week and was underway in all by two weeks. At eight weeks, magnetic resonance imaging and histologic analysis showed deformation of the femoral head and variable tissue deposition. Tissue responses included (1) vascularized fibroblastic ingrowth with tissue resorption and cartilage, intramembranous bone, and mixed fibro-osseous or fibro-cartilaginous tissue synthesis and (2) resumption of endochondral bone growth. At eight weeks, the mean femoral head measurements (and standard error of the mean) for the control compared with the ligated femora were 10.4 +/- 0.4 and 4.8 +/- 0.4 mm, respectively, for height; 26.7 +/- 0.8 and 31.2 +/- 0.8 mm for diameter; 1.1 +/- 0.1 and 2.3 +/- 0.1 mm for cartilage thickness; and 151 degrees +/- 2 degrees and 135 degrees +/- 2 degrees for the femoral neck-shaft angle. Repeated-measures mixed-model analysis of variance revealed highly significant effects of ligation in each parameter (p < 0.0001). CONCLUSIONS: Magnetic resonance imaging allows for the assessment of individual hips at sequential time periods to follow deformation and repair. There was a variable tissue response, and histologic assessment at the time of killing was shown to correlate with the evolving and varying magnetic resonance imaging signal intensities. Femoral head height on the ischemic side from one week onward was always less than the initial control value and continually decreased with time, indicating collapse as well as slowed growth. Increased femoral head width occurred relatively late (four to eight weeks), indicating cartilage model overgrowth concentrated at the periphery.

Calcineurin inhibitors activate the proto-oncogene Ras and promote protumorigenic signals in renal cancer cells.

The development of cancer is a major problem in immunosuppressed patients, particularly after solid organ transplantation. We have recently shown that calcineurin inhibitors (CNI) used to treat transplant patients may play a critical role in the rapid progression of renal cancer. To examine the intracellular signaling events for CNI-mediated direct tumorigenic pathway(s), we studied the effect of CNI on the activation of proto-oncogenic Ras in human normal renal epithelial cells (REC) and renal cancer cells (786-0 and Caki-1). We found that CNI treatment significantly increased the level of activated GTP-bound form of Ras in these cells. In addition, CNI induced the association of Ras with one of its effector molecules, Raf, but not with Rho and phosphatidylinositol 3-kinase; CNI treatment also promoted the phosphorylation of the Raf kinase inhibitory protein and the downregulation of carabin, all of which may lead to the activation of the Ras-Raf pathway. Blockade of this pathway through either pharmacologic inhibitors or gene-specific small interfering RNA significantly inhibited CNI-mediated augmented proliferation of renal cancer cells. Finally, it was observed that CNI treatment increased the growth of human renal tumors in vivo, and the Ras-Raf pathway is significantly activated in the tumor tissues of CNI-treated mice. Together, targeting the Ras-Raf pathway may prevent the development/progression of renal cancer in CNI-treated patients.