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R K Jain

Publications and source records attributed to R K Jain.

At least 37 records · Page 2Linked to original sources
PubMed2002

Solid stress facilitates spheroid formation: potential involvement of hyaluronan.

When neoplastic cells grow in confined spaces in vivo, they exert a finite force on the surrounding tissue resulting in the generation of solid stress. By growing multicellular spheroids in agarose gels of defined mechanical properties, we have recently shown that solid stress inhibits the growth of spheroids and that this growth-inhibiting stress ranges from 45 to 120 mmHg. Here we show that solid stress facilitates the formation of spheroids in the highly metastatic Dunning R3327 rat prostate carcinoma AT3.1 cells, which predominantly do not grow as spheroids in free suspension. The maximum size and the growth rate of the resulting spheroids decreased with increasing stress. Relieving solid stress by enzymatic digestion of gels resulted in gradual loss of spheroidal morphology in 8 days. In contrast, the low metastatic variant AT2.1 cells, which grow as spheroids in free suspension as well as in the gels, maintained their spheroidal morphology even after stress removal. Histological examination revealed that most cells in AT2.1 spheroids are in close apposition whereas a regular matrix separates the cells in the AT3.1 gel spheroids. Staining with the hyaluronan binding protein revealed that the matrix between AT3.1 cells in agarose contained hyaluronan, while AT3.1 cells had negligible or no hyaluronan when grown in free suspension. Hyaluronan was found to be present in both free suspensions and agarose gel spheroids of AT2.1. We suggest that cell-cell adhesion may be adequate for spheroid formation, whereas solid stress may be required to form spheroids when cell-matrix adhesion is predominant. These findings have significant implications for tumour growth, invasion and metastasis.

Animals↗
PubMed2002

Serological and coat protein sequence studies suggest that necrosis disease on sunflower in India is caused by a strain of Tobacco streak ilarvirus.

Serological and coat protein sequence studies were conducted to identify an ilarvirus associated with necrosis disease on sunflower in India. In electroblot immunoassay, sunflower ilarvirus reacted strongly only with antiserum to Tobacco streak virus (TSV). The coat protein gene of sunflower ilarvirus was cloned and sequenced. The sequence analyses also showed that the CP gene was most closely related to TSV, the member of subgroup I of Ilarvirus. The sunflower ilarvirus CP shared 90% amino acid sequence identity with TSV. On the basis of serological relatedness and sequence identity, it is proposed that the sunflower ilarvirus from India should be considered a strain of TSV belonging to subgroup I and designated as TSV-SF. This is the first report of the molecular characterization of TSV on sunflower from the Indian subcontinent.

Antibodies, Viral↗
PubMed2001

Oligomerization of green fluorescent protein in the secretory pathway of endocrine cells.

Green fluorescent protein (GFP) is used extensively as a reporter protein to monitor cellular processes, including intracellular protein trafficking and secretion. In general, this approach depends on GFP acting as a passive reporter protein. However, it was recently noted that GFP oligomerizes in the secretory pathway of endocrine cells. To characterize this oligomerization and its potential role in GFP transport, cytosolic and secretory forms of enhanced GFP (EGFP) were expressed in GH4C1 and AtT-20 endocrine cells. Biochemical analysis showed that cytosolic EGFP existed as a 27 kDa monomer, whereas secretory forms of EGFP formed disulphide-linked oligomers. EGFP contains two cysteine residues (Cys(49) and Cys(71)), which could play a role in this oligomerization. Site-directed mutagenesis of Cys(49) and Cys(71) showed that both cysteine residues were involved in disulphide interactions. Substitution of either cysteine residue resulted in a reduction or loss of oligomers, although dimers of the secretory form of EGFP remained. Mutation of these residues did not adversely affect the fluorescence of EGFP. EGFP oligomers were stored in secretory granules and secreted by the regulated secretory pathway in endocrine AtT-20 cells. Similarly, the dimeric mutant forms of EGFP were still secreted via the regulated secretory pathway, indicating that the higher-order oligomers were not necessary for sorting in AtT-20 cells. These results suggest that the oligomerization of EGFP must be considered when the protein is used as a reporter molecule in the secretory pathway.

Animals↗
PubMed2001

LYVE-1 is not restricted to the lymph vessels: expression in normal liver blood sinusoids and down-regulation in human liver cancer and cirrhosis.

Lymphatic vessel endothelial hyaluronan receptor (LYVE)-1 is thought to be restricted to lymph vessels and has been used as such to show that tumor lymphangiogenesis occurs on overexpression of lymphangiogenic factors in mouse tumor models. However, these studies have not yet been corroborated in human tumors. Here we show, first, that LYVE-1 is not exclusive to the lymph vessels. Indeed, LYVE-1 is also present in normal hepatic blood sinusoidal endothelial cells in mice and humans. Surprisingly, LYVE-1 is absent from the angiogenic blood vessels of human liver tumors and only weakly present in the microcirculation of regenerative hepatic nodules in cirrhosis, though both vessels are largely derived from the liver sinusoids. Second, we propose a novel approach to identify lymphatics in human and murine liver. By combining LYVE-1 and Prox 1 (a transcription factor) immunohistochemistry, we demonstrate that lymphatics are abundant in cirrhosis. In contrast, in human hepatocellular carcinoma and liver metastases, they are restricted to the tumor margin and surrounding liver. The absence of intratumor lymphatics in hepatocellular carcinomas and liver metastases may impair molecular and cellular transport in these tumors. Finally, the presence of LYVE-1 in liver sinusoidal endothelia suggests that LYVE-1 has functions beyond the lymph vascular system.

Animals↗
PubMed2001

Vascular morphogenesis and remodeling in a model of tissue repair: blood vessel formation and growth in the ovarian pedicle after ovariectomy.

To investigate mechanisms of vascular morphogenesis in tissue repair, we performed ovariectomy with resection of the corresponding branches of the ovarian vessels in nude mice. This induces a vascular network remodeling response in the healing ovarian pedicle. Reconstruction of 2000 histological serial sections demonstrated that a new vascular network composed of venous-venous loops forms in the wall of the dilated ovarian vein. Preexisting veins of all sizes, including a branch of the main artery, are subjected to segmentation. Loop formation and segmentation are based on intussusceptive microvascular growth. Loop formation is followed by elongation. Loop remodeling occurs also by intussusception and results in the formation of compound loop systems. All loop systems observed were completely patent. Blind-ending sprouts were extremely rare. Anastomoses between the preexisting vessels subjected to segmentation and the loop systems were established to include the newly formed vessels into the preexisting vascular network. The formation of an increasing number of patent loop systems likely decreases hypoxia and subsequently arrests angiogenesis with transformation of the granulation tissue into a scar. Loop formation also occurred inside a large thrombus that occluded a part of the lumen of the main vein.

Animals↗
PubMed2001

Vascular morphogenesis and remodeling in a human tumor xenograft: blood vessel formation and growth after ovariectomy and tumor implantation.

To determine mechanisms of blood vessel formation and growth in solid tumors, we used a model in which LS174T human colon adenocarcinomas are grown in the isolated ovarian pedicle of nude mice. Reconstruction of 3500 histological serial sections demonstrated that a new vascular network composed of venous-venous loops of varying sizes grows inside the tumor from the wall of the adjacent main vein. Loops elongate and remodel to establish complex loop systems. The mechanisms of loop formation and remodeling correspond to intussusceptive microvascular growth (IMG). In the tissue surrounding the tumor segmentation, another mechanism of IMG is prevalent in venous vessels. Comparison to vascular morphogenesis in the ovariectomized pedicle not only confirms the existence of corresponding mechanisms in both systems, but also reveals numerous sprouts that are superimposed onto loop systems and pathological deviations of loop formation, remodeling, and segmentation in the tumor. These pathological mechanisms interfere with vessel patency that likely cause heterogenous perfusion and hypoxia thus perpetuating angiogenesis. Blood vessel formation based on IMG was also detected in a large thrombus that completely occluded a part of an ovarian artery branch.

Adenocarcinoma↗
PubMed2001

Hypoxia and acidosis independently up-regulate vascular endothelial growth factor transcription in brain tumors in vivo.

Hypoxia and acidosis are hallmarks of tumors as well as critical determinants of response to treatments. They can upregulate vascular endothelial growth factor (VEGF) in vitro. However, the relationship between tissue oxygen partial pressure (pO(2))/pH and VEGF transcription in vivo is not known. Thus, we developed a novel in vivo microscopy technique to simultaneously measure VEGF promoter activity, pO(2), and pH. To monitor VEGF expression in vivo, we engineered human glioma cells that express green fluorescent protein (GFP) under the control of the VEGF promoter. These cells were implanted into the cranial windows in severe combined immunodeficient mice, and VEGF promoter activity was assessed by GFP imaging. Tissue pO(2) and pH were determined by phosphorescence quenching microscopy and ratio imaging microscopy, respectively. These techniques have allowed us to show, for the first time, that VEGF transcription in brain tumors is independently regulated by the tissue pO(2) and pH. One week after tumor implantation, significant angiogenesis was observed, with increased GFP fluorescence throughout the tumor. Under hypoxic or neutral pH conditions, VEGF-promoter activity increased, with a decrease in pO(2) and independent of pH. Under low pH or oxygenated conditions, VEGF-promoter activity increased, with a decrease in pH and independent of pO(2). In agreement with the in vivo findings, both hypoxia and acidic pH induced VEGF expression in these cells in vitro and showed no additive effect for combined hypoxia and low pH. These results suggest that VEGF transcription in brain tumors is regulated by both tissue pO(2) and pH via distinct pathways.

Acidosis↗
PubMed2001

Delivery of molecular medicine to solid tumors: lessons from in vivo imaging of gene expression and function.

Extraordinary advances in molecular medicine and biotechnology have led to the development of a vast number of anti-cancer therapeutic agents. To reach cancer cells in a tumor, a blood-borne therapeutic molecule, particle or cell must make its way into the blood vessels of the tumor and across the vessel wall into the interstitium and finally migrate through the interstitium. Unfortunately, tumors often develop in ways that hinder each of these steps. Our research goals are to analyze each of these steps experimentally and theoretically and then integrate the resulting information in a unified theoretical framework. This paradigm of analysis and synthesis has allowed us to obtain a better understanding of microcirculatory barriers in solid tumors and to develop novel strategies to exploit and/or to overcome these barriers for improved cancer detection and treatment.

Animals↗
PubMed2001

Role of tumor-host interactions in interstitial diffusion of macromolecules: cranial vs. subcutaneous tumors.

The large size of many novel therapeutics impairs their transport through the tumor extracellular matrix and thus limits their therapeutic effectiveness. We propose that extracellular matrix composition, structure, and distribution determine the transport properties in tumors. Furthermore, because the characteristics of the extracellular matrix largely depend on the tumor-host interactions, we postulate that diffusion of macromolecules will vary with tumor type as well as anatomical location. Diffusion coefficients of macromolecules and liposomes in tumors growing in cranial windows (CWs) and dorsal chambers (DCs) were measured by fluorescence recovery after photobleaching. For the same tumor types, diffusion of large molecules was significantly faster in CW than in DC tumors. The greater diffusional hindrance in DC tumors was correlated with higher levels of collagen type I and its organization into fibrils. For molecules with diameters comparable to the interfibrillar space the diffusion was 5- to 10-fold slower in DC than in CW tumors. The slower diffusion in DC tumors was associated with a higher density of host stromal cells that synthesize and organize collagen type I. Our results point to the necessity of developing site-specific drug carriers to improve the delivery of molecular medicine to solid tumors.

Animals↗
PubMed2001

Vascular endothelial growth factor (VEGF)-C differentially affects tumor vascular function and leukocyte recruitment: role of VEGF-receptor 2 and host VEGF-A.

Unlike vascular endothelial growth factor (VEGF)-A, the effect of VEGF-C on tumor angiogenesis, vascular permeability, and leukocyte recruitment is not known. To this end, we quantified in vivo growth and vascular function in tumors derived from two VEGF-C-overexpressing (VC+) and mock-transfected cell lines (T241 fibrosarcoma and VEGF-A-/- embryonic stem cells) grown in murine dorsal skinfold chambers. VC+ tumors grew more rapidly than mock-transfected tumors and exhibited parallel increases in tumor angiogenesis. Furthermore, VEGF-C overexpression elevated vascular permeability in T241 tumors, but not in VEGF-A-/- tumors. Surprisingly, unlike VEGF-A, VEGF-C did not increase leukocyte rolling or adhesion in tumor vessels. Administration of VEGF receptor (VEGFR)-2 neutralizing antibody DC101 reduced vascular density and permeability of both VC+ and mock-transduced T241 tumors. These data suggest that VEGFR-2 signaling is critical for tumor angiogenesis and vascular permeability and that VEGFR-3 signaling does not compensate for VEGFR-2 blockade. An alternate VEGFR, VEGFR-1 or neuropilin-1, may modulate adhesion of leukocytes to tumor vessels.

Animals↗
PubMed2001

Delivery of molecular and cellular medicine to solid tumors.

To reach cancer cells in a tumor, a blood-borne therapeutic molecule or cell must make its way into the blood vessels of the tumor and across the vessel wall into the interstitium, and finally migrate through the interstitium. Unfortunately, tumors often develop in ways that hinder each of these steps. Our research goals are to analyze each of these steps experimentally and theoretically, and then integrate the resulting information in a unified theoretical framework. This paradigm of analysis and synthesis has allowed us to obtain a better understanding of physiological barriers in solid tumors, and to develop novel strategies to exploit and/or to overcome these barriers for improved cancer detection and treatment.

Animals↗
PubMed2001

Predominant role of endothelial nitric oxide synthase in vascular endothelial growth factor-induced angiogenesis and vascular permeability.

Nitric oxide (NO) plays a critical role in vascular endothelial growth factor (VEGF)-induced angiogenesis and vascular hyperpermeability. However, the relative contribution of different NO synthase (NOS) isoforms to these processes is not known. Here, we evaluated the relative contributions of endothelial and inducible NOS (eNOS and iNOS, respectively) to angiogenesis and permeability of VEGF-induced angiogenic vessels. The contribution of eNOS was assessed by using an eNOS-deficient mouse, and iNOS contribution was assessed by using a selective inhibitor [l-N(6)-(1-iminoethyl) lysine, l-NIL] and an iNOS-deficient mouse. Angiogenesis was induced by VEGF in type I collagen gels placed in the mouse cranial window. Angiogenesis, vessel diameter, blood flow rate, and vascular permeability were proportional to NO levels measured with microelectrodes: Wild-type (WT) > or = WT with l-NIL or iNOS(-/-) > eNOS(-/-) > or = eNOS(-/-) with l-NIL. The role of NOS in VEGF-induced acute vascular permeability increase in quiescent vessels also was determined by using eNOS- and iNOS-deficient mice. VEGF superfusion significantly increased permeability in both WT and iNOS(-/-) mice but not in eNOS(-/-) mice. These findings suggest that eNOS plays a predominant role in VEGF-induced angiogenesis and vascular permeability. Thus, selective modulation of eNOS activity is a promising strategy for altering angiogenesis and vascular permeability in vivo.

Animals↗
PubMed2001

Comparative sorting of neuroendocrine secretory proteins: a search for common ground in a mosaic of sorting models and mechanisms.

Endocrine, neuroendocrine and exocrine cells store regulated secretory proteins in secretory granules, while constitutive and constitutive-like secretory proteins are secreted directly without storage. Sorting of secretory proteins takes place in the trans-Golgi network (sorting for entry) or immature secretory granules (sorting by retention). The relative contribution of these sorting steps and the sorting signals and mechanisms involved in each step has been the subject of intense studies and debate in recent years. New evidence now suggests that: (1) two proteins with structurally similar sorting signals can use different sorting mechanisms; (2) one protein with multiple sorting signals can be sorted differently in different cell types; and (3) one cell type can recognize different sorting signals and use different sorting mechanisms. The latter finding suggests that sorting must be a regulated event. While the current image of sorting is complex, recent findings are pointing to common features that form a mosaic of related sorting mechanisms.

Animals↗
PubMed2001

Vascular endothelial growth factor receptor-2-blocking antibody potentiates radiation-induced long-term control of human tumor xenografts.

Antiangiogenic therapy can enhance radiation-induced tumor growth inhibition. However, the effects of combined antiangiogenic and radiation therapy on long-term tumor control and normal tissue response have not been reported. We treated mice bearing two different human tumor xenografts with anti-vascular endothelial growth factor receptor-2 antibody (DC101) and five dose fractions of local radiation and followed them for at least 6 months. DC101 significantly decreased the dose of radiation necessary to control 50% of tumors locally. The decrease was 1.7- and 1.3-fold for the moderately radiosensitive small cell lung carcinoma 54A and the highly radioresistant glioblastoma multiforme U87, respectively. In contrast to tumors, no increase in skin radiation reaction by the antibody was detected. Surprisingly, 44% of mice bearing 54A tumor developed clear ascites after DC101 treatment at its highest dose; this was fatal to 20% of mice. This adverse effect was seen only in mice that received whole-body irradiation 1 day before tumor implantation. The encouraging results on two human tumor xenografts suggest that vascular endothelial growth factor receptor-2 blockade merits further investigation to assess its potential as an enhancer of radiation therapy in the clinic.

Angiogenesis Inhibitors↗
PubMed2001

Kinetics of placenta growth factor/vascular endothelial growth factor synergy in endothelial hydraulic conductivity and proliferation.

Vascular endothelial growth factor (VEGF) was originally discovered as vascular permeability factor because of its ability to increase microvascular permeability to plasma proteins. Since then, it has been shown to induce proliferation and migration in endothelial cells. Placenta growth factor (PlGF) is a member of the VEGF family of growth factors, but has little or undetectable mitogenic activity on endothelial cells. Intriguingly, however, PlGF was able to potentiate the action of low concentrations of VEGF on endothelial cell growth and macromolecule permeability in vitro. Here we show that PlGF can potentiate the effects of VEGF on the hydraulic conductivity of certain endothelial cells and that the duration of pretreatment with PlGF determines the resulting response. Hydraulic conductivity (Lp) was calculated from the water flux across the monolayer of human umbilical vein endothelial cells (HUVECs) or bovine aortic endothelial cells (BAECs). After 2 h of exposure to VEGF(165), the Lp of BAEC monolayers increased threefold, but the Lp of HUVEC monolayers did not increase. PlGF alone induced a small (63%) increase in Lp in BAECs, but not in HUVECs. BAEC, but not HUVEC, monolayers exposed first to PlGF and then to VEGF exhibited a seven- to eightfold increase in Lp. This enhancement in BAEC Lp could be observed for 4 h after the administration of PlGF. PlGF also potentiated the effect of VEGF on BAEC proliferation. Thus, augmentation of VEGF action by PlGF depends on the duration of PlGF exposure and on the origin of endothelial cells.

Animals↗
PubMed2001

Decorin inhibits endothelial migration and tube-like structure formation: role of thrombospondin-1.

Interactions between endothelial cell receptors and the extracellular matrix (ECM) play a critical, yet poorly understood role in angiogenesis. Based on the anti-adhesive role of decorin, we hypothesized that decorin binding to ECM molecules such as thrombospondin-1 (TSP-1) plays a regulatory role in endothelial tube-like structure (TLS) formation. To test this hypothesis, endothelial cells were plated on TSP-1, decorin, or mixed substrates of TSP-1 plus decorin. TLS formation was induced by applying type I collagen on the confluent endothelial monolayer. Cartilage decorin inhibited the formation of TLSs in a concentration-dependent manner. On substrates of high decorin concentrations (2.5 and 5.0 microg/cm(2)) the reduction in TLSs was due either to a reduction in the number of adhering cells or to decreased cell migration. At low decorin concentrations (0.05 and 0.25 microg/cm(2)) the reduction in TLSs was independent of the number of attached cells. Time-lapse video microscopy revealed that decorin substrates facilitated homotypic aggregation and isolated cord formation at the expense of endothelial migration and TLS formation. Consistent with the reduced migration, endothelial cells formed fewer vinculin-positive focal adhesions and actin-stress fibers on decorin substrates. Endothelial migration and TLS formation were also significantly inhibited by skin decorin and the protein core of cartilage decorin. The inhibition of TLS formation by the protein core of cartilage decorin was potentiated by TSP-1. These findings suggest that decorin alone or in combination with TSP-1 interferes with the activation of endothelial cell receptors by ECM molecules, thus blocking intracellular signals that induce cytoskeletal reorganization, migration, and TLS formation.

Cartilage↗