Invited Symposium: Cerebral Artery Pharmacology and Physiology


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Lessons from Recombinant Endothelial Nitric Oxide Synthase Gene Transfer to Cerebral Arteries

Contact Person: Alex F.Y. Chen (chen.alex@mayo.edu)


Nitric oxide (NO) plays an essential role in the regulation of vascular tone of both cerebral and peripheral beds (Faraci and Heistad, 1998; Cooke and Dzau, 1997). A number of vascular diseases have been shown to be associated with an impaired vascular NO production and/or activity, including subarachnoid hemorrhage-induced cerebral vasospasm, arteriosclerosis, thrombosis, diabetes, and hypertension (Cooke and Dzau, 1997).

The field of vascular gene transfer has been rapidly developing in the past decade, and important advances have been made in vector technology, transgene expression efficacy, and site-specific gene targeting (Verma and Somia, 1997; Blau and Khavari, 1997; Kay et al., 1997). Recent studies of eNOS gene transfer and expression in the cerebral vasculature are of considerable importance in advancing our understanding of eNOS transgene expression and function, and represent potential therapeutic strategies for the vascular disorders associated with a deficiency of NO production and/or activity (Chen et al., 1998a, 1998b). In this article, the most recent advances of eNOS gene transfer studies in cerebrovascular bed are summarized and their significance in the study of cerebral vascular biology and disease are discussed.

Nitric Oxide Synthase in the Regulation of Cerebrovascular Tone
NO is a potent endogenous vasodilator. It is synthesized from the guanidino nitrogens of L-arginine through a process that consumes five electrons and results in the formation of co-product L-citrulline by a family of nitric oxide synthases (NOS). The process involves the transfer of electrons between five co-factors including flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), tetrahydrobiopterin (BH4), heme, and calmodulin (CaM), and requires three co-substrates including L-arginine, nicotinamide adenine dinucleotide phosphate (NADPH), and molecular oxygen (O2) (Stuehr, 1997).

Three isoforms of NOS, encoded by three distinct genes on different chromosomes, have been isolated and purified. Both the neuronal (nNOS) and endothelial (eNOS) isoforms are constitutively activated and expressed upon calcium-calmodulin binding following an increase in intracellular calcium. The inducible isoform (iNOS) is activated upon immunological stimulation independent of calcium. All three NOS isoforms are present in cerebral vascular beds, and the activation of the soluble guanylate cyclase with the production of cyclic 3',5'-guanosine monophosphate (cGMP) is one of the primary mechanisms for NO-induced cerebral vasodilation (Faraci and Heistad, 1998).

NO is a simple diatomic molecule that exerts many biological effects in cerebral vascular biology including mediation of vasodilatation, inhibition of platelet aggregation, leukocyte and platelet adhesion, and smooth muscle cell proliferation (Cooke and Dzau, 1997; Faraci and Heistad, 1998).

Cerebrovascular Gene Transfer
Cerebrovascular gene transfer refers to the introduction of genes into relevant cells of intracranial blood vessel wall. It holds significant promise as a tool for studying gene expression and regulation in vascular biology, and as a therapeutic means of controlling local vascular function under diseased conditions (Heistad and Faraci, 1996; Chen et al., 1998a, 1998b). In animal studies to date, the most commonly used methods of peripheral vascular gene delivery include 1) delivering vectors to a surgically isolated segment of vessel in which the side branches have been ligated, or 2) catheter-based delivery to the vessel in vivo. In contrast to gene transfer to the peripheral vasculature, gene delivery to the cerebral vessels has unique problems. A segment of cerebral artery cannot be occluded in order to allow localized gene delivery due to problems with brain ischemia. One potential approach to transduction of the cerebral vasculature would be to deliver genes of interest to the perivascular adventitia via the cerebrospinal fluid (CSF) (Heistad and Faraci, 1996).

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Vascular Gene Transfer Vectors

In recent years, both viral and non-viral vectors have been used for vascular gene transfer studies. Viral vectors currently used for vascular gene transfer include adenovirus, adeno-associated virus, retrovirus, and most recently, lentivirus (Verma and Somia, 1997). These recombinant viruses are genetically modified to be replication-incompetent, and they contain an inserted cDNA sequence of interest (e.g. LacZ, eNOS, etc.) driven by an appropriate promoter (e.g. CMV, RSV, etc.). Non-viral vectors that have been studied in vascular gene transfer include liposomes and molecular conjugates for receptor-mediated gene delivery. Viral vectors generally have higher efficiency in transgene expression but are also more immunogenic than non-viral vectors. Comparisons of these vectors with major advantages and disadvantages for vascular gene transfer have recently been reviewed (Verma and Somia, 1997; Chen et al., 1998b).

For gene transfer to the vascular system, recombinant adenoviruses are the most efficient means of transfer (Schneider and French, 1993; Hitt, et al., 1997). Human adenoviruses are non-enveloped, double-stranded linear DNA viruses of approximately 36 kilobase pairs in size (Hitt, et al., 1997). The viruses enter the host cells via an endocytosis process through the interaction between viral penton complex and their cell surface receptor named CAR (see below). Upon entry into the cell, the viruses are taken up into endosomes, which are then disrupted by the virus, resulting in viral DNA release into the cytoplasm. The viral DNA then enters the nucleus, where it is not incorporated into the host chromosome but remains episomal. The niche of adenoviral vectors for vascular gene transfer includes its ability to transduce both dividing and non-dividing cells with high efficiency ex vivo and in vivo, and the ability to generate high titer stock vector (i.e. up to 1012 pfu/ml). The major drawbacks of first generation adenoviral vectors include the capsid protein-induced, cell-mediated (i.e. CD8+ T cell) immune response which may limit the duration of transgene expression and prevent repeated administration of the vector (Hitt, et al., 1997). The newly developed third generation adenoviral vectors deleted of all viral DNA with the use of cell type specific promoters for specific vascular cell targeting may eventually overcome these problems.

Many studies have examined the use of adenoviral vectors to transduce the vessel wall. Much of the recent work concerning gene transfer to cerebral arterial wall in vivo has utilized a vector encoding b-galactosidase marker gene, to demonstrate the feasibility of adenoviral-mediated gene transfer and to optimize the system. Construction of Recombinant Adenoviral Vector Encoding eNOS Gene We have generated a recombinant adenoviral vector encoding an eNOS gene, driven by the cytomegalovirus (CMV) immediate early promoter, through homologous recombination techniques (Spector and Samaniego, 1995; Chen et al., 1997a). In brief, a full length human serotype 5 wild adenovirus was rendered replication-incompetent by the deletion of the early 1 (E1) region of the virus, with the replacement of a cDNA sequence of the bovine aortic endothelial cell eNOS gene construct. The vector was propagated at high titers on 293 cells, a human embryonic kidney carcinoma helper cell line that expresses the E1 region in trans thus enabling the virus to replicate.

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eNOS Gene Transfer to Cerebral Arteries

We have recently demonstrated that adenoviral vector-mediated eNOS gene transfer to canine cerebral arteries ex vivo resulted in functional transgene expression in the adventitia and endothelium, leading to increased basal production of cGMP with a subsequent reduction in UTP-induced vasoconstriction and enhancement in endothelium-dependent relaxation (Chen et al., 1997a). These findings suggest that cerebral arterial tone can be modulated by recombinant eNOS expression in the vessel wall. More recently, we have successfully delivered adenoviral vectors encoding eNOS gene and b-galactosidase reporter gene into canine cerebral blood vessels in vivo via CSF, by means of vector injection into the cisterna magna (Chen et al., 1997b). Transgene expression are localized in the adventitial fibroblasts of major cerebral arteries, as shown by electron microscopy immunogold labeling (Chen et al., 1997b). In eNOS gene- but not b-galactosidase reporter gene-transduced cerebral arteries, bradykinin-induced relaxation was significantly augmented. The change in vasoreactivity was also accompanied by increased cGMP production (Chen et al., 1997b). These results suggest that perivascular eNOS gene delivery via CSF and functional expression is a feasible approach that does not require interruption of cerebral blood flow.

eNOS Gene Transfer and Cerebral Vasospasm
In vivo functional expression of recombinant eNOS gene in cerebral blood vessels with increased local NO production may have important clinical implications. Subarachnoid hemorrhage-induced cerebral vasospasm has been shown to be associated with an impaired L-arginine-nitric oxide-cGMP pathway (Katusic et al., 1993; Kim et al., 1988, 1992), including a decrease in eNOS mRNA level (Hino et al., 1996) and loss of NOS immunoreactivity (Pluta et al., 1996). Experimental vasospasm could be alleviated by intravenous administration of glycerol trinitrate (Frazee et al., 1981), a well-known nitrovasodilator that releases NO intracellularly, intracarotid NO infusion (Afshar et al., 1995), or restoration of endogenous NO production in arterial wall by administration of L-arginine and superoxide dismutase (Kajita et al., 1993; 1994).

The administration of an adenoviral vector via CSF with functional expression of recombinant eNOS in cerebral arteries raises the possibility of providing continuous NO supply to the underlying smooth muscle cells, and may become a potentially feasible therapeutic strategy in alleviating this devastating complication of subarachnoid hemorrhage. Cerebral vasospasm following subarachnoid hemorrhage occurs between 4 and 12 days after subarachnoid hemorrhage (Macdonald, 1997; Brown and Wiebers, 1998). It is a transient phenomenon, probably due to stimulation of the cerebral blood vessels by blood present in the CSF, which is associated with high morbidity and mortality. Adenoviral vector-mediated transfer of eNOS gene may be useful in this setting as transient transgene expression would be an advantage. Intracranial delivery and functional expression of recombinant eNOS gene in the cerebral vasculature, therefore, may provide a novel and feasible approach for the treatment of certain cerebrovascular diseases such as vasospasm.

Consistent with this concept, adenoviral-mediated reporter gene expression (i.e. b-galactosidase) to the cerebral vessels has recently been demonstrated in the presence of cisternal blood, following administration of the vector to the CSF (Muhonen et al., 1997). More recently, we have shown that recombinant eNOS gene can be successfully expressed in cerebral arteries after subarachnoid hemorrhage in a canine model (Onoue et al., 1998).

Adenoviral Receptor and Heterogeneity of Transgene Expression
Recently, a receptor common for coxsackie B virus and adenovirus serotypes 2 and 5 has been identified by molecular cloning and named CAR (Bergelson et al., 1997; Tomko et al., 1997). The receptor is a 46-kD transmembrane glycoprotein consisting of 365 amino acids. Sequence analysis reveals that it belongs to the immunoglobulin gene superfamily with two extracellular immunoglobulin-like domains, and the deduced amino acid sequences are highly homologous between human and mouse CAR (i.e. 91%) (Bergelson et al., 1997; Tomko et al., 1997). Moreover, the human serotype 2 adenoviral receptor is mapped to chromosome 21 (Mayr and Freimuth, 1997). Transfection of CAR cDNA in cell lines without native CAR has resulted in the expression of functional CAR, as manifested by marked b-galactosidase expression after adenoviral-mediated gene transfer in these cells (Bergelson et al., 1997; Tomko et al., 1997).

The identification of CAR as a functional receptor for adenovirus and its tissue distribution is likely to have great significance in targeted gene delivery to specific tissues. Northern blot analysis has indicated that CAR mRNA is strongly expressed in some human tissues (e.g. heart, brain, pancreas, etc.), while the expression in other tissues is weak (e.g. liver, lung) or absent (e.g. kidney, placenta, skeletal muscle, spleen, etc.) (Bergelson et al., 1997; Tomko et al., 1997). These findings suggest that the efficacy of transgene expression mediated by adenovirus may be related to the degree of CAR expression in different tissues and cells. Indeed, the lack of receptor CAR has been shown to contribute to the resistance of adenoviral infection of ciliated airway epithelial cells as a target for gene therapy of cystic fibrosis (Zabner et al., 1997; Pickles et al., 1998). We have also shown that heterogeneity exists between cerebral and peripheral arteries for recombinant eNOS expression that may reflect differential CAR expression in these vessels (Tsutsui et al., 1998). On the other hand, our recent studies have indicated that both CAR mRNA and protein can be detected in human cerebral arteries, and that adenoviral-mediated gene transfer to human pial arteries is feasible (Chen et al., 1998c).

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Future Directions

The feasibility of transferring eNOS genes to cerebral vasculature has now been demonstrated both in vivo and ex vivo as reviewed in this article. However, a number of problems need to be solved before the technology of cerebral gene transfer can enter clinical arena. The main difficulty is the limitations of currently available gene transfer vectors. Improvements in vector design are currently under intensive investigation, and newer generation vectors are already being tested. Another formidable problem is the technical issue of how to deliver the vectors to the cerebral vessel wall in clinical practice. Therefore, in addition to improvements in vector design, enhanced methods of intracranial gene delivery will be required prior to clinical application of this technology. In addition, future studies on the regulation and manipulation of viral receptor expression will likely provide insights on adenoviral-mediated gene expression that will facilitate efficient vascular gene transduction for human gene therapy.

The authors were supported by U.S. National Institutes of Health grant HL-53524 (Z.S.K.), NIH Research Service Award GM08288 (A.F.Y.C.), American Heart Association/Minnesota Affiliate Grant-in-Aid (A.F.Y.C. and Z.S.K.), Mayo Clinic Harold W. Siebens Molecular Medicine Research Award (A.F.Y.C. and Z.S.K.), and funds from the Bruce and Ruth Rappaport Program in Vascular Biology (Z.S.K.) and the Mayo Foundation.

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Chen, AFY; Zvonimir S. Katusic, ; (1998). Lessons from Recombinant Endothelial Nitric Oxide Synthase Gene Transfer to Cerebral Arteries. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/laher/chen0349/index.html
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