Impact of endostatin gene therapy on myeloid-derived suppressor cells from a metastatic renal cell carcinoma.

AIM: To evaluate the role of endostatin (ES) gene therapy on myeloid-derived suppressor cells (MDSC) in a metastatic model of renal cell carcinoma (RCC). MATERIALS AND METHODS: Balb/C mice bearing orthotopic Renca tumors were treated with NIH/3T3-LendSN or, as a control, with NIH/3T3-LXSN cells. At the end of in vivo experiment, plasma and tissue lung samples were collected. Plasma ES and granulocyte colony stimulating factor (G-CSF) levels were measured by ELISA and Milliplex, respectively. Quantification of CD11b+Gr-1+ cells and their subsets was performed by flow cytometry. Reactive oxygen species (ROS) production was measured in CD11b+Gr-1+ MDSC using the DCFDA marker by flow cytometry. RESULTS: Metastatic RCC (mRCC) induced expansions of CD11b+Gr-1+ MDSC and promoted accumulation of these cells and their subtypes in lymphoid organ and metastases. ES treatment promoted low G-CSF plasmatic levels which were produced by the tumor microenvironment, reflecting the reduced metastatic accumulation of CD11b+Gr-1+ MDSC in the lungs. However, the therapy was selective for granulocytic cells, thus reducing the production of ROS. CONCLUSION: These findings confirm the expansion of MDSC during metastatic progression of RCC and indicate the important role of ES in reducing MDSC and possible use of ES therapy in combined anticancer treatment.

Improving the therapeutic potential of endostatin by fusing it with the BAX BH3 death domain.

Endostatin (ES) inhibits angiogenesis, reducing tumor growth in animal models. However, it has low therapeutic effect in human clinical trials. BAX is a member of the BCL-2 family of proteins; its proapoptotic (BH3) domain interacts with other members of the family in the cytoplasm, to induce apoptosis. Here, we fused the BAX BH3 domain with murine ES, to enhance ES potency. Endothelial cells specifically internalize the fusion protein ES-BAX. The presence of the BAX domain enhances endothelial cell death by apoptosis by 1.8-fold and diminishes microvessel outgrowth in the rat aortic ring assay by 6.5-fold. Daily injections of 15 µg of ES-BAX/g in tumor-bearing mice reduce tumor weight by 86.9% as compared with ES-treated animals. Co-immunoprecipitation assays confirmed that ES-BAX interacts with members of the BCL-2 family. Also, ES interacts with BCL-2, BCL-XL, and BAK in endothelial cell lysates, suggesting a potential new mechanism for the apoptosis induction by ES. The superiority of the ES-BAX antiangiogenic effect indicates that this fusion protein could be a promising therapeutic alternative to treat cancer.

Endostatin gene therapy stimulates upregulation of ICAM-1 and VCAM-1 in a metastatic renal cell carcinoma model.

One of the greatest challenges in urological oncology is renal cell carcinoma (RCC), which is the third leading cause of death in genitourinary cancers. RCCs are highly vascularized and respond positively to antiangiogenic therapy. Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. In this study, we examined the potential of ES-based antiangiogenic therapy to activate tumor-associated endothelial cells in metastatic RCC (mRCC). Balb/c-bearing Renca cells were treated with NIH/3T3-LendSN or, as a control, with NIH/3T3-LXSN cells. The T-cell subsets and lymphocyte populations of tumors, mediastinal lymph nodes and the spleen were assessed by flow cytometry. The expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was assessed by real-time PCR, flow cytometry and immunohistochemistry analysis. ES gene therapy led to an increase in the percentage of infiltrating CD4-interferon (IFN)-γ cells (P<0.05), CD8-IFN-γ cells (P<0.01) and CD49b-tumor necrosis factor-α cells (P<0.01). In addition, ES therapy caused an increase at the mRNA level of ICAM-1 (1.4-fold; P<0.01) and VCAM-1 (1.5-fold) (control vs treated group; P<0.001). Through flow cytometry, we found a significant increase in the CD34/ICAM-1 cells (8.1-fold; P<0.001) and CD34/VCAM-1 cells (1.6-fold; P<0.05). ES gene therapy induced a significant increase in both T CD4 and CD8 cells in the lymph nodes and the spleen, suggesting that ES therapy may facilitate cell survival or clonal expansion. CD49b cells were also present in increased quantities in all of these organs. In this study, we demonstrate an antitumor inflammatory effect of ES in an mRCC model, and this effect is mediated by an increase in ICAM-1 and VCAM-1 expression in tumor-associated endothelial cells.

Collagen XVIII/endostatin expression in experimental endotoxemic acute renal failure

Acute renal failure (ARF) is a frequent complication of Gram-negative sepsis, with a high risk of mortality. Lipopolysaccharide (LPS)-induced ARF is associated with hemodynamic changes that are strongly influenced by the overproduction of nitric oxide (NO) through the cytokine-mediated up-regulation of inducible NO synthase. LPS-induced reductions in systemic vascular resistance paradoxically culminate in renal vasoconstriction. Collagen XVIII is an important component of the extracellular matrix expressed in basement membranes. Its degradation by matrix metalloproteases, cathepsins and elastases results in the formation of endostatin, claimed to have antiangiogenic activity and to be a prominent vasorelaxing agent. We evaluated the expression of endostatin/collagen XVIII in an endotoxemic ARF model. ARF was induced in C57BL/6 mice by intraperitoneal injection of LPS (10 mg/kg) followed by sacrifice 4 and 12 h later. Kidney tissue was the source of RNA and protein and the subject of histological analysis. As early as 4 h after LPS administration, blood urea, creatinine and NO levels were significantly increased compared to control. Endostatin/collagen XVIII mRNA levels were 0.71 times lower than sham-inoculated mice 4 h after LPS inoculation, returning to normal levels 12 h after LPS inoculation. Immunohistological examination revealed that acute injury caused by LPS leads to an increase of endostatin basement membrane staining in association with the decrease of CD31 endothelial basement membrane staining. These results indicate that in the early phase of endotoxemic ARF the endostatin levels were not regulated by gene expression, but by the metabolism of collagen XVIII.

Collagen XVIII/endostatin expression in experimental endotoxemic acute renal failure.

Acute renal failure (ARF) is a frequent complication of Gram-negative sepsis, with a high risk of mortality. Lipopolysaccharide (LPS)-induced ARF is associated with hemodynamic changes that are strongly influenced by the overproduction of nitric oxide (NO) through the cytokine-mediated up-regulation of inducible NO synthase. LPS-induced reductions in systemic vascular resistance paradoxically culminate in renal vasoconstriction. Collagen XVIII is an important component of the extracellular matrix expressed in basement membranes. Its degradation by matrix metalloproteases, cathepsins and elastases results in the formation of endostatin, claimed to have antiangiogenic activity and to be a prominent vasorelaxing agent. We evaluated the expression of endostatin/collagen XVIII in an endotoxemic ARF model. ARF was induced in C57BL/6 mice by intraperitoneal injection of LPS (10 mg/kg) followed by sacrifice 4 and 12 h later. Kidney tissue was the source of RNA and protein and the subject of histological analysis. As early as 4 h after LPS administration, blood urea, creatinine and NO levels were significantly increased compared to control. Endostatin/collagen XVIII mRNA levels were 0.71 times lower than sham-inoculated mice 4 h after LPS inoculation, returning to normal levels 12 h after LPS inoculation. Immunohistological examination revealed that acute injury caused by LPS leads to an increase of endostatin basement membrane staining in association with the decrease of CD31 endothelial basement membrane staining. These results indicate that in the early phase of endotoxemic ARF the endostatin levels were not regulated by gene expression, but by the metabolism of collagen XVIII.

Increased elastic microfibrils and thickening of fibroblastic nuclear lamina in canine cutaneous asthenia.

Cutaneous asthenia is a hereditary connective tissue disease, primarily of dogs and cats, resembling Ehlers-Danlos syndrome in man. Collagen dysplasia results in skin hyperextensibility, skin and vessel fragility, and poor wound healing. The purpose of this study was to describe the histological findings in a dog with a collagenopathy consistent with cutaneous asthenia. An 8-month-old crossbreed female dog presented with lacerations and numerous atrophic and irregular scars. The skin was hyperextensible and easily torn by the slightest trauma. Ultrastructurally, the dermis was comprised of elaunin and oxytalan microfibrils. These are immature fibres in which the fibrillar component is increased but elastin is reduced. Collagen fibres were profoundly disorganized. The fibrils had a highly irregular outline and a corroded appearance when viewed in cross-section, and were spiralled and fragmented in a longitudinal view. Dermal fibroblasts displayed a conspicuous thickening of the nuclear lamina. Nuclear lamins form a fibrous nucleoskeletal network of intermediate-sized filaments underlying the inner nuclear membrane. Mutations in lamins or lamin-associated proteins cause a myriad of genetic diseases collectively called laminopathies. Disruption of the nuclear lamina seems to affect chromatin organization and transcriptional regulation of gene expression. A common link among all laminopathies may be a failure of stem cells to regenerate mesenchymal tissue. This could contribute to the connective tissue dysplasia seen in cutaneous asthenia.

High-yield purification of biosynthetic human growth hormone secreted in Escherichia coli periplasmic space.

A six-step, high-yield purification procedure for the preparation of clinical grade recombinant human growth hormone (rhGH) secreted in bacterial periplasmic space is described. Particular emphasis is given to hormone recovery yields and maximum contaminant host cell elimination. The strategy adopted, in addition to using one precipitation and five chromatographic steps in a particularly efficient sequence, was also based on running E. coli proteins - immunoradiometric assay profiles right after each chromatographic elution. Thus, an overall rhGH recovery higher than 40%, with a final concentration of E. coli proteins below 10 ppm is described for the first time. The accuracy of hGH and total protein quantification, especially in the early steps of the process, and the maximum elimination of hGH-related forms were also studied in detail. For these purposes size-exclusion and reversed-phase HPLC were found to be extremely valuable analytical tools.

Ultrasensitive in vivo bioassay detects bioactive human growth hormone in transduced primary human keratinocytes.

An improved in vivo body weight gain bioassay for the potency determination of human growth hormone (hGH) has been set up in "little" mice (lit/lit), a mutant derived from the C57BL/6J strain. This improved assay now has a detection limit of the order of 0.05 micrograms/mouse/day, which corresponds to a sensitivity about 20-fold higher than that of the most sensitive in vivo assay reported up to now: the tibia test in hypophysectomized rats or mice. This sensitivity was achieved mainly by introduction of a careful pre-assay selection and of a three injections per day schedule. The utilization of these conditions in a 2x2 factorial assay design allowed the potency determination of recombinant DNA-derived hGH (rec-hGH) in bacterial extracts with acceptable accuracy and precision, together with the greatest economy of material, only 0.24 mg of unknown and standard hormone preparation being sufficient for an entire 10-animal assay. This contrasts to a minimum of 2.7 mg that are necessary for an economical assay in hypophysectomized rats. The same assay procedure was also used to demonstrate the in vivo bioactivity of hGH secreted into a culture medium from transduced human primary keratinocytes. The growth curve constructed with n = 8 little mice presented a highly significant correlation (r = 0.939, p < 0.001) and a slope = 0.016 g/mouse/day. It was thus possible to prove, for the first time, the in vivo bioactivity of rec-hGH secreted by transplantable human epidermal cells, utilized as an experimental model for somatic gene therapy.

In vivo bioassay for the potency determination of human growth hormone in dwarf "little" mice.

Homozygous 40- to 90-day-old "little" mice (lit/lit), derived from the C57BL/6J strain, have been used to set up an in vivo body weight gain bioassay for GH whose performance has been compared to the widely used hypophysectomized rat assay. A log dose-response curve has been analyzed in order to choose doses in the linear range that are suitable for setting up useful, precise, and economical 2 x 2 factorial assays. A comparison between the response in the two sexes has also been carried out, showing no significant difference between male and female little mice of this age. Three assays have been carried out in these animals for the potency determination of a local human GH (hGH) reference preparation in terms of First International Standard of GH (human) for bioassay (WHO 80/505), comparing them to a classical assay in hypophysectomized rats performed with the same preparations. The calculated potency values were in good agreement, while the statistical parameters indicated a comparable assay precision, even in a practical and rapid 4-day assay. We suggest the substitution of hypophysectomized rats with little mice for this in vivo test, still required for potency and bioidentity determination of clinical preparations of recombinant hGH. This avoids the highly invasive, costly, and time-consuming surgery, providing a faster, more flexible, and economical assay method, while still directly measuring parameters of in vivo linear growth in an animal model closely related to isolated hGH deficiency type I.