Intracellular barriers to non-viral gene transfer

Non-viral vector mediated gene transfer, compared to viral vector mediated one, is a promising tool for the safe delivery of therapeutic DNA in genetic and acquired human diseases. Although the lack of specific immune response favor the clinical application of non-viral vectors, comprising of an expression cassette complexed to cationic liposome or cationic polymer, the limited efficacy and short duration of transgene expression impose major hurdles in the widespread application of non-viral gene therapy. The trafficking of transgene, complexed with chemical vectors, has been the subject of intensive investigations to improve our understanding of cellular and extracellular barriers impeding gene delivery. Here, we review those physical and metabolic impediments that account, at least in part, for the inefficient translocation of transgene into the nucleus of target cells. Following the internalization of the DNA-polycation complex by endocytosis, a large fraction is targeted to the lysosomal compartment by default. Since the cytosolic release of heterelogous DNA is a prerequisite for nuclear translocation, entrapment and degradation of plasmid DNA in endo-lysosomes constitute a major impediment to efficient gene transfer. Only a small fraction of internalized plasmid DNA penetrates the cytoplasm. Plasmid DNA encounters the diffusional and metabolic barriers of the cytoplasm, further decreasing the number of intact plasmid molecules reaching the nuclear pore complex (NPC), the gateway of nucleosol. Nuclear translocation of DNA requires either the disassembly of the nuclear envelope or active nuclear transport via the NPC. Comparison of viral and plasmid DNA cellular trafficking should reveal strategies that viruses have developed to overcome those cellular barriers that impede non-viral DNA delivery in gene therapy attempts.     

Possibilities of non-viral gene transfer to improve cutaneous wound healing

Enhancement of dermal and epidermal regeneration represents a crucial goal for the treatment of acute, e.g. burn and trauma wounds, and chronic wounds, e.g. diabetic, autoimmune, arterial and venous wounds. Studies defining molecular mechanisms of the complex cascade of wound healing have shown that growth factors represent a new therapeutic strategy. The clinical application of growth factors in the form of proteins has been shown to be of little benefit. Therefore new delivery systems and therapeutic strategies needed to be developed to improve dermal and epidermal regeneration, one of which is gene therapy. For successful gene delivery the selection of an appropriate vector has been shown to be paramount. Because Retroviruses, Adenoviruses and Adeno-Associated Viruses can cause immunologic reactions and mutations, non-viral delivery systems for gene therapy, such as liposomal gene transfer appear advantageous over viral gene therapy. This review discusses the success, potential and limitations of non-viral gene transfer to improve regeneration of dermal and epidermal structures.     

Poly(N-isopropylacrylamide) co-polymer films as potential vehicles for delivery of an antimitotic agent to vascular smooth muscle cells.

INTRODUCTION: Local delivery of antimitotic agents is a potential therapeutic strategy for protection of injured coronary vasculature against intimal hyperplasia and restenosis. This study sought to establish the principle that thermoresponsive poly(N-isopropylacrylamide) co-polymer films can be used to deliver, in a controlled manner, an antimitotic agent to vascular smooth muscle cells (VSMC). METHODS: A series of co-polymer films was prepared, using varying ratios (w/w) of N-isopropylacrylamide (NiPAAm) monomer to N-tert-butylacrylamide (NtBAAm) and loaded with the antimitotic agent colchicine (100 nmol/film) at room temperature. RESULTS: The extent of colchicine release at 37 degrees C was inversely proportional to the amount of NtBAAm in co-polymer films: release after 48 h from 85:15, 65:35 and 50:50 (NiPAAm:NtBAAm) films was 26, 17 and 0.5 nmol, respectively. In cytotoxicity studies, when medium incubated with co-polymers for 24 h (in the absence of colchicine) was further incubated with target bovine aortic smooth muscle cells (BASMC), no loss of cell viability occurred. Colchicine released from all three co-polymer films significantly inhibited proliferation and random migration of BASMC: 100 nM colchicine (released from 65:35 NiPAAm:NtBAAm) reduced cell proliferation to 25.7+/-1.7% of levels seen in the absence of colchicine (control) and random cell migration to 37.7+/-5.7% of control (mean+/-S.E.M., n = 3, P < .01 and P < .05, respectively). The magnitudes of these effects were comparable to those seen in separate experiments with native colchicine and were observed in samples of released colchicine which had been stored at -20 degrees C for up to 6 months. CONCLUSIONS: This study has shown that the release of the antimitotic agent colchicine, from NiPAAm/NtBAAm co-polymer films can be manipulated by changes in co-polymer composition. Furthermore, such drug released at 37 degrees C retains comparable bioactivity to that of native colchicine.     

Cytokeratins in smooth muscle cells and smooth muscle tumours

A keratin positive metastatic leiomyosarcoma in the lung, which resulted in diagnostic error, is reported. The results of additional studies of 17 benign and malignant leiomyogenic tumours with various keratin antibodies are presented and discussed in the light of recent bibliographical data.     

Use of muscle cells to mediate gene transfer to the bone defect.

Segmental bone defects and nonunions are relatively common problems facing all orthopaedic surgeons. Osteogenic proteins, i.e., BMP-2, can promote bone healing in segmental bone defects. However, a large quantity of the human recombinant protein is needed to enhance the bone healing potential. Cell mediated gene therapy in the bone defect can allow a sustained expression of the osteogenic proteins and further enhance bone healing. Muscle cells can be easily isolated and cultivated, and they are known to be an efficient gene delivery vehicle to muscle and nonmuscle tissues. Furthermore, they are capable of transforming into osteoblasts when stimulated by BMP-2. Thus, the utilization of muscle cells as the gene delivery vehicle to a bone defect would be an important step in establishing a less invasive treatment for non-unions and segmental bone defects. Muscle cells were transduced when the adenoviral-lacZ vector and injected into the bone defect and the muscles surrounding the defect. Expression of the marker gene was visualized 7 days after the injection, both macroscopically and microscopically, using lacZ histochemistry. The lacZ expressing cells in the defect tissue were also stained for desmin, a muscle specific marker, indicating the presence of muscle cells that have fused into myofibers in this nonmuscle bone defect area. With successful myoblast mediated gene delivery into the segmental bone defect, future experiments would focus on delivering viral vectors expressing osteogenic proteins to eventually improve bone healing postinjury.     

Plasmid size influences chitosan nanoparticle mediated gene transfer to chondrocytes

The objective of the present study was to prepare chitosan nanoparticles incorporating a relatively large plasmid encoding for osteogenic protein (OP)-1 and to determine the ability of these nanoparticles to transfect adult canine articular chondrocytes in vitro. The positive charge of chitosan acted to condense the relatively large negatively-charged OP-1 plasmid such that it could be incorporated into nanoparticles. Incorporation of the plasmid into the chitosan nanoparticles did not affect the structural integrity of the plasmid as demonstrated by gel electrophoresis. The morphology and size of the nanoparticles were found to vary with the chitosan:plasmid weight ratio. Nanoparticles formulated with a chitosan:plasmid ratio of 10:1 were of uniformly small size (less than 250 nm) and spherical shape. These nanoparticles had a positive charge of about 20 mV. FITC-labeled chitosan nanoparticles were found in virtually all of the cells after 24 h of incubation with the nanoparticles, and confocal microscopy revealed FITC-related fluorescence in the nucleus of the chondrocytes. Although transfection of the chondrocytes was demonstrated by the fluorescence of cells treated with chitosan nanoparticles containing the plasmid for the enhanced green fluorescence protein, cells transfected with nanoparticles incorporating the larger OP-1 plasmid did not show OP-1 expression measured by ELISA for up to 2 weeks in culture. These results indicate that although a large plasmid can be successfully incorporated within chitosan nanoparticles, the size of the plasmid incorporated within the nanoparticles may still significantly affect gene transfer to cells.     

A novel transgenic marker for migrating limb muscle precursors and for vascular smooth muscle cells.

A unique pattern of LacZ expression was found in a transgenic mouse line, likely due to regulatory elements at the site of integration. Two new genes flanking the transgene were identified. At early stages of development, the transgene is transiently expressed in ventro-lateral demomyotomal cells migrating from the somites into the limb buds. At late developmental stages and in the adult, lacZ staining marks vascular smooth muscle cells throughout the vascular bed, with the exception of the major elastic arteries, and in pericytes. No expression was detected in skeletal and smooth muscles. Different patterns of expression in vascular smooth muscles was observed at distinct levels of the vascular tree, in arteries as well as in veins. Vessel injury, resulting in stimulation of smooth muscle cells proliferation and migration, is associated with transgene down-regulation. After the formation of neointima thickening, it is reactivated. This transgenic insertion may therefore be used as a useful marker to identify novel physiological cues or genetic elements involved in the regulation of the vascular smooth muscle phenotype(s). It may also provide an experimental tool for studying vasculature and the involvement of pericytes in regulating microvascular homeostasis.     

人脐动脉平滑肌细胞培养及转染TFPI基因的实验研究

Objective The aim of the present study is to investigate the effect of tissue factor pathway inhibitor (TFPI) gene on cell cycle of human vascular smooth muscle cells.Methods Human vascular smooth muscle cells were separated from human umbilical artery and identified by immunohistochemical staining.The cells were transfected with various amount of pIRES-TFPI plasmid (1,2,and 3 μg/ml,respectively)and the TFPI expression in the cells were analyzed by RT-PCR.MTT assay was employed to detect the effect of TFPI gene on the proliferation of human vascular smooth muscle cells.Results The proliferation of vascular smooth muscle cells was inhibited in pIRES-TFPI group 5 and 7 days after gene transfection when compared with that of pIRES 1-neo transfection group.Conclusion The overexpression of TFPI gene in human umbilical artery vascular smooth muscle cells may contribute to the suppression of the proliferation of cells by gene transfection.     

人脐动脉平滑肌细胞培养及转染TFPI基因的实验研究

目的 探讨组织因子途径抑制因子（TFPI）基因对人脐动脉血管平滑肌细胞生长的影响,为TFPI基因用于血管再狭窄的治疗提供理论依据和实验基础.方法 从人脐动脉分离平滑肌细胞,通过免疫组化方法进行细胞鉴定;用不同剂量pIRES-TFPI基因（分别为1,2,3 μg/mL）转染血管平滑肌细胞,采用RT-PCR测定细胞内TFPI表达以优化基因转染条件;通过MTT法测定TFPI基因对人脐动脉血管平滑肌细胞生长的影响.结果 分离得到的血管平滑肌细胞的纯度高于90%;3个剂量的基因转染后,细胞内TFPI基因的表达水平无明显差异.采用2 μg/mL转染剂量时,TFPI基因转染后第5天,脐动脉血管平滑肌的生长受到明显抑制.结论 通过基因转染的方式将TFPI基因导入细胞对人脐动脉平滑肌的增殖具有抑制作用.     

Cytocompatibility studies of an in situ photopolymerized thermoresponsive hydrogel nanoparticle system using human aortic smooth muscle cells

We have been investigating thermoresponsive hydrogel nanoparticle composite networks to develop photopolymerized hydrogels to deliver drugs for prevention of restenosis after angioplasty. These composite systems can form a gel under physiological conditions and release drugs in response to temperature changes. Our novel system, consisting of poly(N-isopropylacrylamide) thermoresponsive nanoparticles, photo cross-linker poly(ethylene glycol) diacrylate, and UV photoinitiator, 2-hydroxy-1-[4-(2-hydroxyethoxy) phenyl]-2-methyl-1-propanone-1-one (Irgacure 2959), would be photopolymerized in situ in the presence of UV light. The focus of this study was to evaluate the effects of photoinitiator and UV exposure on human aortic smooth muscle cells (HASMCs). We found that the exposure to UV light did not significantly affect the cellular survival within doses required for photopolymerization. The photoinitiator was cytocompatible at low concentrations (< or = 0.015% w/v); however, cytotoxicity increased with increasing photoinitiator concentrations. In addition, free radicals formed in the presence of a photoinitiator and UV light caused significant levels of cell death. An antioxidant (free radical scavenger), ascorbic acid, added to the cell media, significantly improved relative cell survival but increased the hydrogel gelation time. Finally, HASMC survival when exposed to all potential cytotoxic components was also evaluated by exposing HASMCs to media incubated with our composite hydrogels. In summary, our studies show that the photoinitiator and free radicals are responsible for the cytotoxicity on HASMCs, and the addition of antioxidants can significantly reduce these harmful effects.     

A system for the direct co-culture of endothelium on smooth muscle cells

The development of a functional, adherent endothelium is one of the major factors limiting the successful development of tissue engineered vascular grafts (TEVGs). The adhesion and function of endothelial cells (ECs) on smooth muscle cells (SMCs) are poorly understood. The goal of this research was to optimize conditions for the direct culture of endothelium on SMCs, and to develop an initial assessment of co-culture on EC function. The co-culture consisted of a culture substrate, a basal adhesion protein, a layer of porcine SMCs, a medial adhesion protein, and a layer of porcine ECs. Conditions that led to successful co-culture were: a polystyrene culture substrate, a quiescent state for SMCs, subconfluent density for SMC seeding and confluent density for EC seeding, and fibronectin (FN) for the basal adhesion protein. EC adhesion was not enhanced by addition of FN, collagen I, collagen IV or laminin (LN) to the medial layer. 3-D image reconstruction by confocal microscopy indicated that SMCs did not migrate over ECs and the cells were present in two distinct layers. Co-cultures could be consistently maintained for as long as 10 days. After exposure to 5 dyne/cm(2) for 7.5 h, ECs remained adherent to SMCs. PECAM staining indicated junction formation between ECs, but at a lower level than that observed with EC monocultures. Co-culturing ECs with SMCs did not change the growth rate of ECs, but EC DiI-Ac-LDL uptake was increased. Thus, a confluent and adherent layer of endothelium can be directly cultured on quiescent SMCs.     

A novel system for investigating the ability of smooth muscle cells and fibroblasts to regulate adhesion of flowing leukocytes to endothelial cells

Allergic contact dermatitis is a frequent and increasing health problem. For ethical reasons, the current animal tests used to screen for contact sensitizers should be replaced by in vitro alternatives. Contact sensitizers have been shown to accelerate Langerhans cell (LC) migration from human organotypic skin explant cultures (hOSECs) more rapidly than non-sensitizers and it has been proposed that the hOSEC model could be used to screen for sensitizers. However, chemically induced decreases in epidermal LC numbers need to be accurately quantified if the alterations in epidermal LC numbers are to form the basis of an alternative system for screening contact sensitizers in vitro. As manual counting of LCs is labour intensive and subject to intra- and inter-personal variation we developed an image analysis routine, using the Leica QWin image analysis software, to quantify LCs in situ using immunohistochemically stained skin sections. LCs can be identified using antibodies against the membrane molecule CD1a or the Lag antibody, which recognises cytoplasmic Birbeck granules. Quantification of epidermal LC number using the image analysis software had a much lower inter-person variation than when the same specimens were counted manually, using both the anti-Lag and CD1a antibodies. The software-aided quantification of epidermal LCs provides an accurate method for measuring chemically-induced changes in LC numbers.     

Chlamydia pneumoniae facilitates monocyte adhesion to endothelial and smooth muscle cells

Chlamydia pneumoniae has been linked to atherosclerotic heart disease. However, there is a limited knowledge by which C. pneumoniae gain access to atheromatous lesions. The adhesion of C. pneumoniae -infected circulatory component(s) to endothelium and smooth muscle cells represents the first step in an inflammatory response. We examined the ability of viable as well as heat inactivated C. pneumoniae to infect human monocytes and subsequently the ability of infected monocytes to adhere to human coronary artery endothelial cells (HCAEC) and human coronary smooth muscle cells (HCSMC). Our results demonstrate susceptibility of monocytes to in vitro chlamydial infection. Inclusions of varying sizes and intensities were observed 3-5 days after inoculation with viable C. pneumoniae. Monocytes infected with heat inactivated organisms revealed no inclusions, in keeping with the observations of uninfected monocytes. Moreover, monocytes infected with viable C. pneumoniae adhered preferentially to HCAEC and HCSMC, as compared to uninfected monocytes or monocytes harbouring heat inactivated Chlamydia.     

HSV-1 amplicon vectors are an efficient gene transfer system for skeletal muscle cells

HSV-1 amplicon vectors have been considered as a promising gene delivery system for gene therapy of skeletal muscle diseases, due to the ability to infect non-dividing cells such as differentiated muscle cells, and to accommodate large transgenes such as the 14-kb dystrophin cDNA. Studies revealed that HSV-1 amplicons can transduce cultured differentiated and undifferentiated muscle cells with high efficiency. Studies also revealed that HSV-1 amplicons are capable of delivering at least 23-kb transgene DNA, including the full-length dystrophin cDNA into muscle cells. The combination of high transduction efficiency, the ability to accommodate large constructs and ease of manipulation makes HSV-1 amplicons an ideal gene delivery tool for the study of muscle ion channels in which gene transduction is frequently employed in cultured muscle cells that are resistant to all the transfecting reagents. However, intramuscular injection of HSV-1 amplicons has been proven inefficient in mature muscles. Evidence has shown that this is mainly because the basal membrane that sheaths each myofibers blocks HSV-1 virions from myofiber cell surface receptors. This result led to the conclusion that HSV-1 amplicons are more suitable for ex vivo manipulation of diseased muscle progenitors or stem cells for autologous cell therapy than in vivo intramuscular injection. Efforts to confer stable transduction ability on amplicons have made progress. A new generation of HSV/AAV hybrid amplicons has been shown to be capable of integrating large transgenes into the AAVS1 site of the human genome, thus, holding potential to achieve a safe and lasting gene transduction in human muscle cells.     

A method for isolating and culturing vascular smooth muscle cells from porcine cerebral arteries

Cerebral blood vessels have unique properties when compared to most peripheral vascular beds. One such property is that large cerebral vessels are involved in the regulation of cerebral vascular resistance. Studying smooth muscle cells isolated from these vessels will determine how phenotypic properties of these cells contribute to unique cerebrovascular function. Therefore we developed a method of culturing smooth muscle cells from explants of cerebral arteries of porcine brains obtained, gratis, from a local slaughter house. Cells isolated and cultured by the methods described herein were of smooth muscle origin as indicated by histochemical staining for smooth muscle -actin. Further, we examined the response of the cultured cells to agonists which activate the cGMP dependent vasodilator system by stimulating soluble guanylyl cyclase (nitroglycerin and sodium nitroprusside) or particulate guanylyl cyclase (C-type natriuretic peptide). Also, forskolin activation of adenylyl cyclase was examined. These agents stimulated an increase in intracellular cGMP and cAMP in a manner that was reproducible in every cell isolation (20 brains) and which remained unchanged through nine passages. Additionally, the cells could be frozen, thawed and replated without loss of responsiveness to these agents. The protocol reported here provides a method for culturing cerebral artery smooth muscle cells that is inexpensive, relatively simple, and which yields cells that can be utilized through multiple passages.Abbreviations DMEM Dulbecco's modified Eagle's medium - FBS Fetal bovine serum - HEPES N-2-hydroxyethylpiperizine-N'-2-ethanesulfonic acid - PBS Phosphate buffered saline     

A simple method for differentiating vascular smooth muscle cells and fibroblasts in tissue culture

Summary The morphologic differentiation of vascular smooth muscle cells and fibroblasts in tissue culture is difficult if not impossible. By direct immunofluorescence, it is possible to distinguish between vascular smooth muscle cells and fibroblasts after 6 to 10 days in tissue culture. Microfilaments appear from the 6th to the 10th day. After an incubation period of 30 minutes with antibody against smooth muscle actomyosin at room temperature, microfilaments are demonstrable in smooth muscle cells. In contrast, fibroblasts, if incubated for the same period, show strong nuclear fluorescence and a primary fluorescence of the cytoplasm, but filaments are not visible. If fibroblasts are incubated with antiactomyosin for one hour at 37 °C, however, microfilaments are easily detectable.With this method it is possible to differentiate in a simple manner vascular smooth muscle cells from fibroblasts in a heterologous tissue culture.These studies were supported by the Deutsche Forschungsgemeinschaft, SFB 90,Cardiovasculäres System.     

Direct protein transfer to terminally differentiated muscle cells

Recently, a new approach for direct protein transfer to mammalian cells based on the herpes simplex virus type 1 protein VP22 has been described. This protein has the remarkable property of intercellular trafficking, which is independent of direct cell contacts and is also retained when fused to heterologous proteins. However, the spreading has only been described for proliferating cells and has also been controversially discussed. In this study we describe the generation of a GFP-VP22 fusion protein which is able to spread in COS-7 cells after transient transfection. Moreover, we show in coculture experiments with transfected COS-7 cells and C2C12 myotubes that this fusion protein is also able to spread into terminally differentiated skeletal muscle cells. These results suggest that VP22 might be a novel therapeutic tool for direct protein transfer not only in proliferating but also in terminally differentiated cells.     

转染TFPI基因对人前列腺平滑肌细胞增殖的影响

目的良性前列腺增生（BPH）是严重危害老年男性健康的常见疾病,本研究旨在研究组织因子途径抑制因子（TFPI）基因对前列腺平滑肌细胞生长的影响,为良性前列腺增生的基因治疗提供参考依据。方法取前列腺增生患者手术切除的前列腺组织,采用酶消化法分离前列腺平滑肌细胞;免疫组化方法进行细胞鉴定;pIRES—TFPI基因转染前列腺平滑肌细胞,以pIRES基因作为基因转染阴性对照;用细胞计数法和四氮唑蓝MTT法观察细胞增殖情况;采用RT-PCR检测细胞内TFPI基因的表达情况。结果SMA免疫组化染色和MASSON染色显示：经过5次传代后,前列腺平滑肌细胞的纯度达到95％以上;TFPI基因转染后,前列腺平滑肌细胞内的TFPImRNA水平明显提高,是未转染组的7倍、阴性对照基因转染组的3．5倍;基因转染4d后,TFPI基因转染组的前列腺平滑肌细胞数明显低于阴性对照基因转染组。结论提示TFPI基因对前列腺平滑肌细胞的增殖具有调控作用,有必要对其作用机理进行进一步研究。