VEGF gene delivery to myocardium: deleterious effects of unregulated expression.

BACKGROUND: Vascular endothelial growth factor (VEGF) is being investigated for therapeutic angiogenesis in ischemic myocardium. Primarily, transient delivery systems have been tested. The goal of this study was to investigate the effects of continuous expression of VEGF in myocardium by use of myoblast-mediated delivery. METHODS AND RESULTS: Primary murine myoblasts (5 x 10(5) cells in 10 microL of PBS with 0.5% BSA) expressing both the murine VEGF gene and the beta-galactosidase (beta-gal) gene from a retroviral promoter were implanted in the ventricular wall of immunodeficient mice (n=11) via a subdiaphragmatic approach. Control immunodeficient mice (n=12) were injected with the same number of myoblasts expressing only the beta-gal gene. Between days 14 and 16, surviving mice were euthanized and the hearts processed for histology. In the experimental group, 11 of 11 mice demonstrated failure to thrive by day 13; 5 deaths occurred between days 8 and 15. There were no complications in the control mice. Histochemistry documented successful implantation of myoblasts (positive beta-gal reaction product) in 6 of 6 surviving experimental mice and 12 of 12 controls. Histology disclosed intramural vascular tumors resembling hemangiomas in the VEGF-myoblast-injected myocardium in 6 of 6 surviving mice. beta-Gal-expressing cells were present at the site of the vascular tumors. Immunohistochemistry localized abundant endothelial nitric oxide synthase and CD31 (platelet and endothelial cell adhesion molecule) within the lesion, consistent with the presence of endothelial cells. CONCLUSIONS: In this model, unregulated continuous expression of VEGF is associated with (1) a high rate of failure to thrive/death and (2) formation of endothelial cell-derived intramural vascular tumors in the implantation site. These results underscore the importance of regulating VEGF expression for therapeutic angiogenesis.

Angiogenesis monitored by perfusion with a space-filling microbead suspension.

Numerous laboratories are focusing efforts on delivering gene products to induce or prevent the development of new blood vessels in adults, with the hope of rescuing ischemic tissues, circumventing cardiac bypass surgery, or inhibiting tumor growth. Current approaches to the assessment of vascular continuity involve the introduction of either dyes or fluorescent microspheres to track blood flow. However, dyes and dextrans are subject to leakage when vessels are hyperpermeable, a situation that may occur in studies of tumor vasculature and during efforts to stimulate therapeutic angiogenesis. Furthermore, the microspheres that are used for flow studies do not allow a comprehensive visual analysis of vascular continuity. Here we report a method for the visual assessment of microvascular continuity in mouse muscle under circumstances in which vessels are leaky. The approach involves perfusion of the vasculature with fluorescent beads that are much smaller than those used for flow studies. The suspension behaves like a fluid and completely fills the vessels, yet the beads do not leak from VEGF-permeablized capillaries and remain localized in histological sections. Use of beads with the proper fluorescence emission wavelengths allows immunofluorescent colocalization with vessel-specific markers. We compare this improved method with other methods for tracking vascular continuity involving dextrans and larger beads. This approach should aid in the dynamic study of tumor angiogenesis and the evaluation of efforts to deliver angiogenic factors.

Induction of angiogenesis by implantation of encapsulated primary myoblasts expressing vascular endothelial growth factor.

BACKGROUND: We previously demonstrated that intramuscular implantation of primary myoblasts engineered to express vascular endothelial growth factor (VEGF) constitutively resulted in hemangioma formation and the appearance of VEGF in the circulation. To investigate the potential for using allogeneic myoblasts and the effects of delivery of VEGF-expressing myoblasts to non-muscle sites, we have enclosed them in microcapsules that protect allogeneic cells from rejection, yet allow the secretion of proteins produced by the cells. METHODS: Encapsulated mouse primary myoblasts that constitutively expressed murine VEGF164, or encapsulated negative control cells, were implanted either subcutaneously or intraperitoneally into mice. RESULTS: Upon subcutaneous implantation, capsules containing VEGF-expressing myoblasts gave rise to large tissue masses at the implantation site that continued to grow and were composed primarily of endothelial and smooth muscle cells directly surrounding the capsules, and macrophages and capillaries further away from the capsules. Similarly, when injected intraperitoneally, VEGF-producing capsules caused significant localized inflammation and angiogenesis within the peritoneum, and ultimately led to fatal intraperitoneal hemorrhage. Notably, however, VEGF was not detected in the plasma of any mice. CONCLUSIONS: We conclude that encapsulated primary myoblasts persist and continue to secrete VEGF subcutaneously and intraperitoneally, but that the heparin-binding isoform VEGF164 exerts localized effects at the site of production. VEGF secreted from the capsules attracts endothelial and smooth muscle cells in a macrophage-independent manner. These results, along with our previous results, show that the mode and site of delivery of the same factor by the same engineered myoblasts can lead to markedly different outcomes. Moreover, the results confirm that constitutive delivery of high levels of VEGF is not desirable. In contrast, regulatable expression may lead to efficacious, safe, and localized VEGF delivery by encapsulated allogeneic primary myoblasts that can serve as universal donors.

A novel means of drug delivery: myoblast-mediated gene therapy and regulatable retroviral vectors.

A potentially powerful approach to drug delivery in the treatment of disease involves the use of cells to introduce genes encoding therapeutic proteins into the body. Candidate genes for delivery include those encoding secreted factors that could have broad applications ranging from treatment of inherited single-gene deficiencies to acquired disorders of the vasculature or cancer. Myoblasts, the proliferative cell type of skeletal muscle tissues, are potent tools for stable delivery of a gene of interest into the body, as they become an integral part of the muscle into which they are injected, in close proximity to the circulation. The recent development of improved tetracycline-inducible retroviral vectors allows for fine control of recombinant gene expression levels. The combination of ex vivo gene transfer using myoblasts and regulatable retroviral vectors provides a powerful toolbox with which to develop gene therapies for a number of human diseases.

Analysis of immune responses to varicella zoster viral proteins induced by DNA vaccination.

In this study we sought to examine the mechanism by which immune responses were induced following intramuscular injection of mice with DNA expression vectors encoding genes of varicella zoster virus (VZV). Both VZV-specific antibody and T cell proliferative responses were induced by immunization with DNA sequences for the immediate early 62 (IE62) and glycoprotein E (gE). The viral proteins were shown to be expressed in non-regenerating, rather than regenerating muscle cells. After primary immunization, muscle cells did not express major histocompatibility complex (MHC) class II transcripts and little inflammatory response was detected at the site of inoculation. Histochemical staining and non-isotopic in situ hybridization demonstrated that a second injection of IE62 plasmid DNA was again associated with protein synthesis in non-regenerating muscle cells but that a marked inflammatory infiltrate was induced in muscle tissue. These cells, but not muscle cells, expressed MHC class II transcripts. Significantly, PCR analyses demonstrated that IE62 DNA localized specifically to local draining lymph nodes following primary DNA immunization by intramuscular inoculation. These experiments indicate that transport of plasmid DNA to sites of antigen presentation in regional lymphoid tissue may play an important role in the initial generation of immune responses and that enhancement by secondary inoculation is mediated by immune cells that traffic to the site of viral protein synthesis in muscle cells.

VEGF gene delivery to muscle: potential role for vasculogenesis in adults.

Constitutive expression of VEGF after implantation of genetically engineered myoblasts into non-ischemic muscle led to an increase in vascular structures. Previously, effects of VEGF delivery to adult muscle have only been reported in ischemic tissues. The resulting vascular structures were reminiscent of those formed during embryonic vasculogenesis, rather than angiogenesis, sprouting from preexisting vessels. Initially, VEGF caused an accumulation of endothelial cells and macrophages, followed by networks of vascular channels and hemangiomas with locally high serum VEGF levels. No effects were evident in adjacent tissue or contralateral legs, where low serum VEGF was detected. These data suggest that the induction by VEGF of angiogenesis or vasculogenesis may be dose-dependent. Furthermore, VEGF expression must be carefully modulated, as overexpression is deleterious.

Inhibition of solid tumor growth by Fas ligand-expressing myoblasts.

A major problem with standard treatments of solid tumors such as chemotherapy is that the effects are not localized to the tumor. As a result, normal tissue function is often severely impaired. Here we show that myoblasts from skeletal muscle that have been engineered with retroviral vectors to express Fas ligand (FasL) have potential as site-specific anti-tumor agents. FasL-expression by myoblasts was previously shown to lead to neutrophil-mediated immunodestruction, both of the cells and the surrounding tissue. Moreover, myoblasts expressing FasL induced apoptosis in Fas-expressing human tumor cells in vitro. These findings led us to investigate the possibility that myoblasts expressing FasL could serve as anti-tumor agents acting by both apoptotic and immunological mechanisms. The C57BL/6 lpr/lpr mouse primary myoblasts either expressing or not expressing murine FasL were co-injected with Fas-positive or Fas-negative human rhabdomyosarcoma cells into the tibialis anterior of immunodeficient mice. After 19-31 days, FasL-expressing myoblasts resulted in a marked accumulation of neutrophils and inhibited tumor growth in every case. By contrast, control myoblasts did not prevent significant tumor growth. The status of Fas expression by the tumor tissue in vivo was confirmed by immunostaining tumor sections with antibodies against Fas. Tumor inhibition was observed regardless of the presence or absence of Fas on the tumor cells, suggesting that in vivo, the induction of a neutrophil response is remarkably potent and sufficient to inhibit tumors.

The effects of testicular tissue and prehatching inhibition of estrogen synthesis on the development of courtship and copulatory behavior in zebra finches.

As in many mammalian and avian species, testicular androgens or their metabolites activate courtship and copulatory behaviors in adult male zebra finches. However, studies of sexual differentiation of these behaviors and related anatomical structures provide conflicting results. For example, posthatching estradiol can both masculinize courtship and the neural structures involved in song in females and inhibit the development of masculine copulation in males. These and other results have led to the hypotheses that (1) testicular androgens are converted to estradiol in the brain of developing males, and estradiol serves to masculinize the song system, whereas (2) estradiol secretion by the female ovary allows feminine rather than masculine copulatory behavior to develop. Treating embryonic zebra finches with the estrogen synthesis inhibitor fadrozole causes functional testicular tissue to develop in genetic females. The present study investigated the effects of such treatment on the development of singing and copulatory behavior as well as song system anatomy in males and females. While exogenous testosterone facilitated the display of sexual behaviors in adult males, the testicular tissue in females had no masculinizing effect on the production of audible courtship song or copulation. Their song control nuclei were also not masculinized, even in individuals lacking ovarian tissue. In contrast, embryonic inhibition of estrogen synthesis in males significantly stimulated song production. These results suggest that while manipulations of steroid hormone exposure can influence the display of sexual behaviors, gonadal secretions may not be required for normal sexual differentiation of the song system in zebra finches.

High-efficiency retroviral infection of primary myoblasts.

In the past, it has been hard to introduce genes into primary myoblasts without selection, as they have been very difficult to transfect or infect. We describe conditions under which mouse primary skeletal muscle myoblasts can be infected with retroviral vectors at high efficiency. Infection can be greatly increased by minimizing the time during which cells are exposed to virus, adding a minimal centrifugation step, and supplementing the infection cocktail to mimic more closely primary myoblast growth medium. Under these conditions, one round of exposure to virus results in an infection efficiency of up to 80%, whereas 4-5 rounds of infection over a two day period reproducibly yield an infection efficiency of > 99%. These methods greatly enhance the potential for studying genetically engineered primary myoblasts from any mouse strain, transgenic or knockout, and may have useful application to other primary cell types that are refractory to transfection or infection.

Neither testicular androgens nor embryonic aromatase activity alters morphology of the neural song system in zebra finches.

A variety of sexual dimorphisms exist in zebra finches, both in the central nervous system and in the periphery. For example, male zebra finches sing to court females, whereas females do not normally sing. In parallel, the brain regions that control song contain a variety of male-biased sexual dimorphisms, and the vocal organ (syrinx) is larger in males than in females. It is thought that some, if not all, of these sex differences are caused by differential hormone secretion by the testes and ovaries of developing birds. However, the effects on sexual differentiation of altering the hormonal environment of birds after hatching have been ambiguous. We have found that treating embryonic female zebra finches with fadrozole, a potent aromatase inhibitor, can induce testicular tissue to develop in addition to normal ovarian tissue. This study documents the function of that testicular tissue in females by showing that it secretes androgens in adulthood, produces sperm, and causes the androgen-sensitive syrinx to enlarge. However, that functional testicular tissue has no effect on the neural song system of females; the brain regions remain completely feminine. Therefore, these results suggest that endocrinologically active testicular tissue alone is not responsible for the dramatic morphological sex differences in the zebra finch song system.

Stage-specific requirement for myosin II during Dictyostelium development.

Dictyostelium cells that lack a functional myosin II heavy chain are motile and are capable of aggregation, but fail to undergo further multicellular development. We have used a Dictyostelium mutant expressing a cold-sensitive myosin heavy chain to examine the requirement for myosin throughout the course of development. The loss of myosin function upon cooling is rapid and reversible. Temperature-shift experiments reveal that myosin is essential during two different stages of development. During aggregation, myosin function appears to be necessary for cells to sort correctly in a way that allows further development to occur. During the final stage of development, it is required for the formation of a complete stalk and the raising of the spore head. Development between those stages, however, proceeds normally in the absence of myosin function. Aggregates at non-permissive temperature undergo an aberrant form of development resulting in a ball of cells. Calcofluor staining and reporter gene fusions reveal that these structures contain defective spores and a miniature stalk.

Genetic control of fungal differentiation: the three sporulation pathways of Neurospora crassa.

Sporulation in the mold Neurospora crassa can proceed along three very different pathways, leading to the production of three types of spores. Two asexual sporulation pathways that lead to the formation of macroconidia and microconidia involve budding from hyphae by two different mechanisms. A much more complex sexual reproductive pathway involves the formation of a fruiting body called a perithecium, in which meiosis takes place and ascospores are formed in sac-like cells called asci. Numerous mutations exist that affect these developmental pathways and genes have been isolated that are expressed preferentially during sporulation. The Neurospora sporulation pathways offer a simple system with which to study mechanisms and regulation of development that are usually obscured by complex cell-cell interactions involved in animal and plant development.

Timing of synthesis and cellular localization of two conidiation-specific proteins of Neurospora crassa.

The process of conidiation in Neurospora crassa consists of a series of distinct developmental stages culminating in the formation of multinucleate asexual spores called macroconidia. Immunoblotting techniques were used to study the timing of synthesis and cellular localization of CON10 and CON13, the products of two genes that are expressed during conidiation but not during mycelial growth. Both proteins first appear about 8 hr into conidiation; CON10 disappears between 2 and 4 hr after germination. Within conidiating cultures, CON10 and CON13 proteins are localized in conidiophores, with little or no protein present in the underlying mycelium. Immunofluorescence analyses show that CON10 is evenly distributed throughout the cytoplasm of macroconidia. Synthesis of CON10 and CON13 occurs at a time when their specifying mRNAs first appear (Hager and Yanofsky, Gene 96, 153-159, 1990; Sachs and Yanofsky, Dev. Biol 148, 117-128, 1991), suggesting that regulation of synthesis is predominantly transcriptional.

Expression of con genes along the three sporulation pathways of Neurospora crassa.

The filamentous fungus Neurospora crassa produces three types of spores by using different developmental pathways: macroconidiation, microconidiation, and sexual spore (ascospore) formation. Several genes of unknown function have been cloned by virtue of their expression during macroconidiation but not during mycelial growth (con genes). It had been postulated that expression of the con genes was specific to macroconidiation. To test this assumption, protein extracts from macroconidia, microconidia, ascospores, and protoperithecia (sexual structures) were analyzed for the product of one of the con genes, con-10, by immunoblotting using a CON10-specific antiserum. CON10 was detected in all of these extracts. An immunologically related protein was detected in an extract from ascospores of a nonconidiating Neurospora species, N. africana. Total RNA isolated from the three types of N. crassa spores was analyzed for con gene mRNA by Northern blotting using five different con genes as probes. Transcripts for four of the genes were detected in all three spore types; mRNA for the fifth gene was detected in macroconidia and microconidia but not in ascospores. Analysis of aconidial and female sterile mutants showed that expression of the con genes along any one developmental pathway occurs when expression along another pathway is genetically blocked.

A morphological and genetic analysis of conidiophore development in Neurospora crassa.

The filamentous fungus Neurospora crassa responds to nutrient deprivation and dessication by producing asexual spores, or conidia. These conidia are derived from differentiated aerial structures called conidiophores. The process of conidiation was analyzed in wild-type and morphological mutants using scanning electron microscopy (SEM) and specific fluorescent probes. The first discernible morphological step of conidiation is the transition from growth by hyphal tip elongation to growth by repeated apical budding, resulting in the formation of chains of proconidia that resemble beads on a string. The initial proconidial chains are morphologically distinct from those that form later and are capable of reverting to hyphal growth, whereas the later chains are committed to conidiation. As the proconidial chains are formed, nuclei migrate into the conidiophore, and cross-walls arise between adjoining proconidia in a series of steps that have been defined by staining with Calcofluor, a fluorescent chitin-binding probe. The chains ultimately disarticulate in several discrete stages into free, morphologically mature conidia. Different conidiation-defective mutants were shown to be blocked at distinct stages in conidiation. Our observations permit us to derive a developmental timeline of conidiation relating the occurrence of morphological changes and the stage blocked in specific mutants.