Enhanced in vivo angiogenesis within a model tissue engineered construct using biodegradable microspheres containing encapsulated VEGF

Introduction. Tissue engineering techniques offer a potential means to develop a tissue engineered construct (TEC) for the treatment of tissue and organ deficiencies. However, a lack of adequate vascularization is a limiting factor in the development of most viable engineered tissues. Vascular endothelial growth factor (VEGF) could aid in the development of a viable vascular network within TECs. The long-term goals of this research are to develop clinically relevant, appropriately vascularized TECs for use in humans. This project tested the hypothesis that the delivery of VEGF via controlled release from biodegradable microspheres would increase the vascular density and rate of angiogenesis within a model TEC. ^ Materials and methods. Biodegradable VEGF-encapsulated microspheres were manufactured using a novel method entitled the Solid Encapsulation/Single Emulsion/Solvent Extraction technique. Using a PLGA/PEG polymer blend, microspheres were manufactured and characterized in vitro. A model TEC using fibrin was designed for in vivo tissue engineering experimentation. At the appropriate timepoint, the TECs were explanted, and stained and quantified for CD31 using a novel semi-automated thresholding technique. ^ Results. In vitro results show the microspheres could be manufactured, stored, degrade, and release biologically active VEGF. The in vivo investigations revealed that skeletal muscle was the optimal implantation site as compared to dermis. In addition, the TECs containing fibrin with VEGF demonstrated significantly more angiogenesis than the controls. The TECs containing VEGF microspheres displayed a significant increase in vascular density by day 10. Furthermore, TECs containing VEGF microspheres had a significantly increased relative rate of angiogenesis from implantation day 5 to day 10. ^ Conclusions. A novel technique for producing microspheres loaded with biologically active proteins was developed. A defined concentration of microspheres can deliver a quantifiable level of VEGF with known release kinetics. A novel model TEC for in vivo tissue engineering investigations was developed. VEGF and VEGF microspheres stimulate angiogenesis within the model TEC. This investigation determined that biodegradable rhVEGF 165-encapsulated microspheres increased the vascular density and relative rate of angiogenesis within a model TEC. Future applications could include the incorporation of microvascular fragments into the model TEC and the incorporation of specific tissues, such as fat or bone. ^

Conditional expression of Sry, and Wnt4 by gene-targeting: Studies in sexual and skeletal development

Sry and Wnt4 cDNAs were individually introduced into the ubiquitously-expressed Rosa26 ( R26) locus by gene targeting in embryonic stem (ES) cells to create a conditional gene expression system in mice. In the targeted alleles, expression of these cDNAs should be blocked by a neomycin resistance selection cassette that is flanked by loxP sites. Transgene expression should be activated after the blocking cassette is deleted by Cre recombinase. ^ To test this conditional expression system, I have bred R26-stop- Sry and R26-stop-Wnt4 heterozygotes with a MisRII-Cre mouse line that expresses Cre in the gonads of both sexes. Analysis of these two types of bigenic heterozygotes indicated that their gonads developed normally like those of wild types. However, one XX R26-Sry/R26-Sry; MisR2-Cre/+ showed epididymis-like structures resembling those of males. In contrast, only normal phenotypes were observed in XY R26-Wnt4/R26-Wnt4; MisR2-Cre /+ mice. To interpret these results, I have tested for Cre recombinase activity by Southern blot and transcription of the Sry and Wnt4 transgenes by RT-PCR. Results showed that bigenic mutants had insufficient activation of the transgenes in their gonads at E12.5 and E13.5. Therefore, the failure to observe mutant phenotypes may have resulted from low activity of MisR2-Cre recombination at the appropriate time. ^ Col2a1-Cre transgenic mice express Cre in differentiating chondrocytes. R26-Wnt4; Col2a1-Cre bigenic heterozygous mice were found to exhibit a dramatic alteration in growth presumably caused by Wnt4 overexpression during chondrogenesis. R26-Wnt4; Col2a1-Cre mice exhibited dwarfism beginning approximately 10 days after birth. In addition, they also had craniofacial abnormalities, and had delayed ossification of the lumbar vertebrate and pelvic bones. Histological analysis of the growth plates of R26-Wnt4; Col2a1-Cre mice revealed less structural organization and a delay in onset of the primary and secondary ossification centers. Molecular studies confirmed that overexpression of Wnt4 causes decreased proliferation and early maturation of chondrocytes. In addition, R26-Wnt4; Col2a1-Cre mice had decreased expression of vascular endothelial growth factor (VEGF), suggesting that defects in vascularization may contribute to the dwarf phenotype. Finally, 9-month-old R26-Wnt4; Col2a1-Cre mice had significantly more fat cells in the marrow cavities of their metaphysis long bones, implying that long-term overexpression of Wnt4may cause bone marrow pathologies. In conclusion, Wnt4 was activated by Col2a1-Cre recombinase and was overexpressed in the growth plate, resulting in aberrant proliferation and differentiation of chondrocytes, and ultimately leads to dwarfism in mice. ^

Correction of aberrant FGFR1 RNA splicing through blocking intronic regulatory elements

Analysis of the human genome has revealed that more than 74% of human genes undergo alternative RNA splicing. Aberrations in alternative RNA splicing have been associated with several human disorders, including cancer. ^ We studied the aberrant expression of alternative RNA splicing isoforms of the Fibroblast Growth Factor Receptor 1 (FGFR1) gene in a human glioblastoma cancer model. Normal glial cells express the FGFR1α, which contains three extracellular domains. In tumors the most abundant isoform is the FGFR1β, which lacks the first extracellular domain due to the skipping of a single exon, termed alpha. The skipping of the α-exon is regulated by two intronic silencing sequences within the precursor mRNA. Since we observed no mutations on these elements in tumor cells, we hypothesized that the over-expression of regulatory proteins that recognize these sequences is responsible for the aberrant expression of splicing isoforms. Hence, we blocked the formation of protein complexes on the ISS using antisense RNA oligonucleotides in vitro. We also evaluated the impact of the ISS antisense oligonucleotides on the endogenous FGFR1 splicing, in a glioblastoma cell model. By targeting intronic regulatory elements we were able to increase the level of alpha exon inclusion up to 90% in glioblastoma cells. The effect was dose dependent, sequence specific and reproducible in glioblastoma and other cancer cells, which also exhibit an alpha exon skipping phenotype. Targeting FGFR1 endogenous ISS1 and ISS2 sequences did not have an additive or synergistic effect, which suggest a regulatory splicing mechanism that requires the interaction of complexes formed on these elements. An increase in the levels of the FGFR1α isoform resulted in a reduction in cell invasiveness. Also, a significant increase in the levels of caspase 3/7 activities, which is indicative of an elevation in apoptosis levels, suggests that expression of FGFR1β might be relevant for tumor survival. These studies demonstrate that it is possible to prevent aberrant expression of exon skipping events through the targeting of intronic regulatory elements, providing an important new therapeutic tool for the correction of human disease caused by alternative RNA splicing. ^

Loss of ovarian function promotes angiogenesis in human ovarian carcinoma

We show here that elevated levels of gonadotropins (luteinizing hormone and follicle stimulating hormone), as found in menopause or after ovariectomy, promote growth of human ovarian carcinoma by induction of tumor angiogenesis. Human epithelial ovarian cancer tumors progressed faster in ovariectomized mice. This induced growth could be attributed to the elevated levels of gonadotropins associated with loss of ovarian function because direct administration of gonadotropins also was effective in promoting tumor progression in vivo. On the other hand, gonadotropins had no direct effect on the proliferation of human ovarian cancer cells in vitro. Using MRI, we demonstrated that ovariectomy significantly (P < 0.02) induces neovascularization of human ovarian carcinoma spheroids implanted in nude mice. Moreover, conditioned medium of gonadotropin-treated human ovarian carcinoma cells showed increased mitogenic activity to bovine endothelial cells, and this activity could be blocked by neutralizing antibodies against luteinizing hormone and against vascular endothelial growth factor. Accordingly, gonadotropin stimulation resulted in a dose-dependent-induced expression of vascular endothelial growth factor in monolayer culture as well as in the outer proliferating cells of human ovarian cancer spheroids. These results demonstrate the significance of the elevated levels of gonadotropins, as found in menopause and in all ovarian cancer patients, on the progression of ovarian cancer and could explain the protective effect of estrogen replacement therapy. Based on these results, we suggest that hormonal therapy aimed at lowering the circulating levels of gonadotropins may possibly prolong remission in ovarian cancer by extending tumor dormancy.

PEG-3, a nontransforming cancer progression gene, is a positive regulator of cancer aggressiveness and angiogenesis

Cancer is a progressive disease culminating in acquisition of metastatic potential by a subset of evolving tumor cells. Generation of an adequate blood supply in tumors by production of new blood vessels, angiogenesis, is a defining element in this process. Although extensively investigated, the precise molecular events underlying tumor development, cancer progression, and angiogenesis remain unclear. Subtraction hybridization identified a genetic element, progression elevated gene-3 (PEG-3), whose expression directly correlates with cancer progression and acquisition of oncogenic potential by transformed rodent cells. We presently demonstrate that forced expression of PEG-3 in tumorigenic rodent cells, and in human cancer cells, increases their oncogenic potential in nude mice as reflected by a shorter tumor latency time and the production of larger tumors with increased vascularization. Moreover, inhibiting endogenous PEG-3 expression in progressed rodent cancer cells by stable expression of an antisense expression vector extinguishes the progressed cancer phenotype. Cancer aggressiveness of PEG-3 expressing rodent cells correlates directly with increased RNA transcription, elevated mRNA levels, and augmented secretion of vascular endothelial growth factor (VEGF). Furthermore, transient ectopic expression of PEG-3 transcriptionally activates VEGF in transformed rodent and human cancer cells. Taken together these data demonstrate that PEG-3 is a positive regulator of cancer aggressiveness, a process regulated by augmented VEGF production. These studies also support an association between expression of a single nontransforming cancer progression-inducing gene, PEG-3, and the processes of cancer aggressiveness and angiogenesis. In these contexts, PEG-3 may represent an important target molecule for developing cancer therapeutics and inhibitors of angiogenesis.

Identification of a Human VPF/VEGF 3′ Untranslated Region Mediating Hypoxia-induced mRNA Stability

Hypoxia is a prominent feature of malignant tumors that are characterized by angiogenesis and vascular hyperpermeability. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) has been shown to be up-regulated in the vicinity of necrotic tumor areas, and hypoxia potently induces VPF/VEGF expression in several tumor cell lines in vitro. Here we report that hypoxia-induced VPF/VEGF expression is mediated by increased transcription and mRNA stability in human M21 melanoma cells. RNA-binding/electrophoretic mobility shift assays identified a single 125-bp AU-rich element in the 3′ untranslated region that formed hypoxia-inducible RNA-protein complexes. Hypoxia-induced expression of chimeric luciferase reporter constructs containing this 125-bp AU-rich hypoxia stability region were significantly higher than constructs containing an adjacent 3′ untranslated region element without RNA-binding activity. Using UV-cross-linking studies, we have identified a series of hypoxia-induced proteins of 90/88 kDa, 72 kDa, 60 kDa, 56 kDa, and 46 kDa that bound to the hypoxia stability region element. The 90/88-kDa and 60-kDa species were specifically competed by excess hypoxia stability region RNA. Thus, increased VPF/VEGF mRNA stability induced by hypoxia is mediated, at least in part, by specific interactions between a defined mRNA stability sequence in the 3′ untranslated region and distinct mRNA-binding proteins in human tumor cells.

Comparative evaluation of the antitumor activity of antiangiogenic proteins delivered by gene transfer

Although the systemic administration of a number of different gene products has been shown to result in the inhibition of angiogenesis and tumor growth in different animal tumor models, the relative potency of those gene products has not been studied rigorously. To address this issue, recombinant adenoviruses encoding angiostatin, endostatin, and the ligand-binding ectodomains of the vascular endothelial growth factor receptors Flk1, Flt1, and neuropilin were generated and used to systemically deliver the different gene products in several different preexisting murine tumor models. Single i.v. injections of viruses encoding soluble forms of Flk1 or Flt1 resulted in ≈80% inhibition of preexisting tumor growth in murine models involving both murine (Lewis lung carcinoma, T241 fibrosarcoma) and human (BxPC3 pancreatic carcinoma) tumors. In contrast, adenoviruses encoding angiostatin, endostatin, or neuropilin were significantly less effective. A strong correlation was observed between the effects of the different viruses on tumor growth and the activity of the viruses in the inhibition of corneal micropocket angiogenesis. These data underscore the need for comparative analyses of different therapeutic approaches that target tumor angiogenesis and provide a rationale for the selection of specific antiangiogenic gene products as lead candidates for use in gene therapy approaches aimed at the treatment of malignant and ocular disorders.

Excessive tumor-elaborated VEGF and its neutralization define a lethal paraneoplastic syndrome

Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen and key regulator of both physiologic and pathologic (e.g., tumor) angiogenesis. In the course of studies designed to assess the ability of constitutive VEGF to block tumor regression in an inducible RAS melanoma model, mice implanted with VEGF-expressing tumors sustained high morbidity and mortality that were out of proportion to the tumor burden. Documented elevated serum levels of VEGF were associated with a lethal hepatic syndrome characterized by massive sinusoidal dilation and endothelial cell proliferation and apoptosis. Systemic levels of VEGF correlated with the severity of liver pathology and overall clinical compromise. A striking reversal of VEGF-induced liver pathology and prolonged survival were achieved by surgical excision of VEGF-secreting tumor or by systemic administration of a potent VEGF antagonist (VEGF-TRAPR1R2), thus defining a paraneoplastic syndrome caused by excessive VEGF activity. Moreover, this VEGF-induced syndrome resembles peliosis hepatis, a rare human condition that is encountered in the setting of advanced malignancies, high-dose androgen therapy, and Bartonella henselae infection. Thus, our findings in the mouse have suggested an etiologic role for VEGF in this disease and may lead to diagnostic and therapeutic options for this debilitating condition in humans.

Negative regulation of hypoxia-inducible genes by the von Hippel-Lindau protein.

Inactivation of the von Hippel-Lindau protein (pVHL) has been implicated in the pathogenesis of renal carcinomas and central nervous system hemangioblastomas. These are highly vascular tumors which overproduce angiogenic peptides such as vascular endothelial growth factor/vascular permeability factor (VEGF/VPF). Renal carcinoma cells lacking wild-type pVHL were found to produce mRNAs encoding VEGF/VPF, the glucose transporter GLUT1, and the platelet-derived growth factor B chain under both normoxic and hypoxic conditions. Reintroduction of wild-type, but not mutant, pVHL into these cells specifically inhibited the production of these mRNAs under normoxic conditions, thus restoring their previously described hypoxia-inducible profile. Thus, pVHL appears to play a critical role in the transduction of signals generated by changes in ambient oxygen tension.

Oligodeoxynucleotides inhibit retinal neovascularization in a murine model of proliferative retinopathy.

Diseases characterized by retinal neovascularization are among the principal causes of visual loss worldwide. The hypoxia-stimulated expression of vascular endothelial growth factor (VEGF) has been implicated in the proliferation of new blood vessels. We have investigated the use of antisense phosphorothioate oligodeoxynucleotides against murine VEGF to inhibit retinal neovascularization and VEGF synthesis in a murine model of proliferative retinopathy. Intravitreal injections of two different antisense phosphorothioate oligodeoxynucleotides prior to the onset of proliferative retinopathy reduced new blood vessel growth a mean of 25 and 31% compared with controls. This inhibition was dependent on the concentration of antisense phosphorothioate oligodeoxynucleotides and resulted in a 40-66% reduction in the level of VEGF protein, as determined by Western blot analysis. Control (sense, nonspecific) phosphorothioate oligodeoxynucleotides did not cause a significant reduction in retinal neovascularization or VEGF protein levels. These data further establish a fundamental role for VEGF expression in ischemia-induced proliferative retinopathies and a potential therapeutic use for antisense phosphorothioate oligodeoxynucleotides.

Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development.

We have recently cloned the human fms-like tyrosine kinase 4 gene FLT4, whose protein product is related to two vascular endothelial growth factor receptors FLT1 and KDR/FLK1. Here the expression of FLT4 has been analyzed by in situ hybridization during mouse embryogenesis and in adult human tissues. The FLT4 mRNA signals first became detectable in the angioblasts of head mesenchyme, the cardinal vein, and extraembryonally in the allantois of 8.5-day postcoitus (p.c.) embryos. In 12.5-day p.c. embryos, the FLT4 signal decorated developing venous and presumptive lymphatic endothelia, but arterial endothelia were negative. During later stages of development, FLT4 mRNA became restricted to vascular plexuses devoid of red cells, representing developing lymphatic vessels. Only the lymphatic endothelia and some high endothelial venules expressed FLT4 mRNA in adult human tissues. Increased expression occurred in lymphatic sinuses in metastatic lymph nodes and in lymphangioma. Our results suggest that FLT4 is a marker for lymphatic vessels and some high endothelial venules in human adult tissues. They also support the theory on the venous origin of lymphatic vessels.

Novelties in Diabetic Retinopathy

Although diabetic retinopathy (DR) remains a leading cause of vision loss, the last decade has brought significant advances in the diagnosis and treatment of this common complication of diabetes mellitus. First, optical coherence tomography allows for noninvasive imaging of the retina, in particular, the macula, with very high resolution, thus facilitating the management of diabetic macular edema. In addition, recent advances in the understanding of the pathophysiology of DR, in particular, the key role of cytokines, such as vascular endothelial growth factor (VEGF), have led to the development of anti-VEGF antibodies for intraocular use. Anti-VEGF therapies have largely replaced laser photocoagulation for the treatment of diabetic macular edema. The benefit of intravitreal anti-VEGF in diabetic macular edema has been proven in numerous large randomized controlled trials. Moreover, a role of inflammation in DR has been recognized, and several mainly steroid-based, anti-inflammatory agents for intravitreal treatment have been shown to be effective. Despite these recent advances, strict systemic control of glycemia remains the cornerstone of the management of DR, significantly reducing ocular complications. This chapter will provide an overview of current and novel concepts of DR and will allude to promising novel therapeutic options for this sight-threatening disease.

Unlocking pathology archives for molecular genetic studies: a reliable method to generate probes for chromogenic and fluorescent in situ hybridization

Chromogenic (CISH) and fluorescent ( FISH) in situ hybridization have emerged as reliable techniques to identify amplifications and chromosomal translocations. CISH provides a spatial distribution of gene copy number changes in tumour tissue and allows a direct correlation between copy number changes and the morphological features of neoplastic cells. However, the limited number of commercially available gene probes has hindered the use of this technique. We have devised a protocol to generate probes for CISH that can be applied to formalin-fixed, paraffin-embedded tissue sections (FFPETS). Bacterial artificial chromosomes ( BACs) containing fragments of human DNA which map to specific genomic regions of interest are amplified with phi 29 polymerase and random primer labelled with biotin. The genomic location of these can be readily confirmed by BAC end pair sequencing and FISH mapping on normal lymphocyte metaphase spreads. To demonstrate the reliability of the probes generated with this protocol, four strategies were employed: (i) probes mapping to cyclin D1 (CCND1) were generated and their performance was compared with that of a commercially available probe for the same gene in a series of 10 FFPETS of breast cancer samples of which five harboured CCND1 amplification; (ii) probes targeting cyclin-dependent kinase 4 were used to validate an amplification identified by microarray-based comparative genomic hybridization (aCGH) in a pleomorphic adenoma; (iii) probes targeting fibroblast growth factor receptor 1 and CCND1 were used to validate amplifications mapping to these regions, as defined by aCGH, in an invasive lobular breast carcinoma with FISH and CISH; and (iv) gene-specific probes for ETV6 and NTRK3 were used to demonstrate the presence of t(12; 15)(p12; q25) translocation in a case of breast secretory carcinoma with dual colour FISH. In summary, this protocol enables the generation of probes mapping to any gene of interest that can be applied to FFPETS, allowing correlation of morphological features with gene copy number.

ANTICANCER MECHANISM OF TOLFENAMIC ACID IN COLORECTAL CANCER

Colorectal cancer (CRC) is the third leading cause of cancer-related death in the United States. Chemopreventive therapies could be effective way to treat CRC. Tolfenamic acid, one of the NSAIDs, shows anti-cancer activities in several types of cancer. Aberrant Wnt/β-catenin regulation pathway is a major mechanism of colon tumorigenesis. Here, we sought to better define the mechanism by which tolfenamic acid suppresses colorectal tumorigenesis focusing on regulation of β-catenin pathway. Treatment of tolfenamic acid led to a down-regulation of β-catenin expression in dose dependent manner in human colon cancer cell lines without changing mRNA. MG132 inhibited tolfenamic acid-induced downregulation of β-catenin and exogenously overexpression β-catenin was stabilized in the presence of tolfenamic acid. Tolfenamic acid induced an ubiquitin-mediated proteasomal degradation of β-catenin. In addition, tolfenamic acid treatment decreased transcriptional activity of β-catenin and expression of Smad2 and Smad3 while overexpression of Smad 2 inhibited tolfenamic acid-stimulated transcriptional activity of β-catenin. Moreover, tolfenamic acid decreased β-catenin target gene such as vascular endothelial growth factor (VEGF) and cyclin D1. In summary, tolfenamic acid is a promising therapeutic drug targeting Smad 2-mediated downregulation of β-catenin in CRC.

Transplantation of artificial gelatin-co-bletillastriata gelatin/Salvia miltiorrhiza Corium promotes dermal repair in rats

Purpose: To evaluate the growth of the composite corium (constructed with fibroblast cells and gelatinco- Bletillastriata gelatin/Salvia miltiorrhiza materials) on rats. Methods: The composite artificial corium was constructed by culturing fibroblast cells in gelatin-co- Bletillastriata gelatin/Salvia miltiorrhiza materials. Full-thickness area of skin was excised from the mice and subsequently, the composite corium was transplanted on the wound. Thereafter, the growth difference of the composite artificial corium and natural corium were compared. In addition, real-time fluorogenic reverse transcription polymerase chain reaction (qRT-PCR) and western blot were performed to determine vascular endothelial growth factor (VEGF) expression at gene and protein levels. Results: The composite artificial corium showed significant repair promoting effect on the skin, and the structure of the repaired skin was similar to that of natural corium. Interestingly, PCR and western blot results showed that the expressions of VEGF were higher in composite artificial corium than in natural corium on days 3 and 7 post-transplantation. Conclusion: The composite artificial corium has some clinical prospects for use in the treatment of wounds on large areas of skin.