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Circulation. 1995;91:521-531

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(Circulation. 1995;91:521-531.)
© 1995 American Heart Association, Inc.

Articles

Genetic Models of Human Vascular Disease

Victor J. Dzau, MD; Gary H. Gibbons, MD; Brian K. Kobilka, MD; Richard M. Lawn, PhD; Richard E. Pratt, PhD

From the American Heart Association–Bugher Foundation Center for Molecular Biology of the Cardiovascular System, Stanford University School of Medicine, Stanford, Calif.

Correspondence to Victor J. Dzau, MD, Falk Cardiovascular Research Center, Stanford University School of Medicine, 300 Pasteur Dr, Stanford, CA 94305-5246.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowInbred Genetic Animal Models...
 down arrowTransgenic Animal Models
down arrowGene Targeting and Knockout...
 down arrowIn Vivo Gene Transfer...
 down arrowConclusions
down arrowReferences
 
Abstract The use of genetic models has greatly assisted investigations of the natural history, mechanisms, and potential therapy for human vascular disease. In the past, genetic models of vascular disease were obtained through serendipity and/or selective breeding to obtain inbred lines that express the phenotype of interest. This approach has yielded several valuable models of atherosclerosis and hypertension. In the past several years, the advent of molecular techniques has enabled investigators to produce additional novel genetic models of disease that have further enhanced the study of vascular biology and medicine. Transgenic techniques and the techniques of homologous recombination have allowed researchers to alter the genotype of an animal in a precise manner and to study the resultant change in phenotype. More recently, techniques of in vivo gene transfer have also accelerated and enhanced the development of novel models. The application of these methodologies has resulted in important breakthroughs in our understanding of the pathogenesis and treatment of vascular diseases. In this review, we compare and contrast these technologies along with examples of their use in the studies of vascular biology and medicine.


Key Words: hypertension • atherosclerosis • genes • recombination • mapping


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowInbred Genetic Animal Models...
 down arrowTransgenic Animal Models
down arrowGene Targeting and Knockout...
 down arrowIn Vivo Gene Transfer...
 down arrowConclusions
down arrowReferences
 
The concept of the American Heart Association–Bugher Foundation Centers for Molecular Biology is built on the premise that future advances in the prevention and treatment of human cardiovascular disease will be fostered by application of the technology of molecular biology to cardiovascular research. The fulfillment of Koch's postulates to define the causal interrelations between genetic and environmental factors as determinants of human vascular diseases has been an elusive goal in cardiovascular medicine. A critical aspect of advancing our understanding of the pathogenesis of human vascular disease involves the integration of clinical observations that have defined the risk factors for cardiovascular disease with the development of animal models of human disease and the characterization of the pathogenic mediators of disease at the molecular level. Recent developments in the technology of molecular genetics have dramatically enhanced our capacity to study the influence of genetic factors on human vascular disease as well as to develop more sophisticated models of human vascular disease that can be used to characterize the molecular basis of disease. The American Heart Association–Bugher Foundation Center at Stanford is actively using novel technologies to study the genetic and molecular basis of vascular disease and to develop novel genetic models. In this review, we focus on hypertension and atherosclerosis as paradigms for illustrating the use of molecular and genetic technology to further elucidate the pathogenesis of human vascular disease. We describe the current approaches to studying the genetic and molecular basis of vascular disease and generating novel genetic models: inbred genetic strains, transgenic animals, gene targeting by homologous recombination, and in vivo gene transfer. In particular, we illustrate the integration between observations defined in humans by medical genetics and new insights gained from the use of molecular genetic analysis and genetic engineering technology in animal models.


*    Inbred Genetic Animal Models
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Inbred Genetic Animal Models...
down arrowTransgenic Animal Models
down arrowGene Targeting and Knockout...
 down arrowIn Vivo Gene Transfer...
 down arrowConclusions
down arrowReferences
 
Until recently, genetic models of vascular disease were obtained through serendipity and breeding. Several genetic models of naturally occurring vascular diseases exist. In hypertension, inbred models of genetic hypertension such as the spontaneously hypertensive rat (SHR), the stroke-prone SHR (SHRSP), the Dahl salt-sensitive rat, and the John Rapp salt-sensitive strain are available for study.1 Furthermore, mouse strains of genetic hypertension are also available. In atherosclerosis, the most notable model is the Watanabe heritable hyperlipidemic (WHHL) rabbit.2 In addition, a variety of outbred rabbits and primates are also prone to atherosclerosis if fed a high-cholesterol diet. Similarly, inbred mouse strains that are predisposed to atherosclerosis have been characterized.3 4 These inbred genetic models are useful for studies defining the pathophysiology of vascular disease. For example, the natural history and pathology of atherosclerosis have been studied extensively in the WHHL rabbit,2 and the information derived from these studies has been extremely useful in understanding human atherosclerosis. Similarly, important alterations in cardiac and vascular regulation and structure that contribute to the development or maintenance of hypertension have also been described in the SHR.1 These include impaired endothelial function, altered angiotensin responsiveness, and vascular and cardiac hypertrophy. However, it is unclear whether any or all of these abnormalities are primary (causative) or secondary (resultant) to genetic hypertension.

The specific genes responsible for hypertension have not been identified. Thus, one of the most valuable contributions of inbred genetic models is the mapping and identification of the genes responsible for this disease. Epidemiological, family, and twin studies suggest that a substantial proportion of the phenotypic variation in blood pressure is genetically determined. These studies also reveal that human essential hypertension does not follow a simple mendelian mode of inheritance but rather is a heterogeneous, polygenic disease with environmental influences. Molecular genetic analysis provides a powerful means of identifying the actual causative genes in hypertension. These studies can determine chromosomal localization of the culprit genes and can lead to the identification and subsequent cloning of these genes. However, despite the recent identification of a linkage between angiotensinogen gene and human hypertension,5 it is generally acknowledged that the genetic analysis of human hypertension is extremely difficult at this time.

The availability of the SHR and other inbred hypertensive animal models offers substantial advantages over studies in humans because of the genetic homogeneity and the large number of offspring available for study. Once the locations of the genes are identified by animal linkage studies, it is possible to find the corresponding region in the human by synteny conservation between regions in animal and human genomes. Once we find the syntenic region, we will be able to screen affected sib pairs and pedigrees for its role in human hypertension, using genetic markers around the region. The specific genes responsible for hypertension in the animal model can then be identified by molecular cloning strategies directed by the chromosomal position.

Using this overall strategy, we and our collaborators began studies to dissect the genes for hypertension by initially crossbreeding SHRSP and the control Wistar-Kyoto (WKY) rats (Fig 1aDown) and subsequently crossbreeding SHR and Brown Norway (BN) rats.6 7 We reported initially that a locus on chromosome 10 is linked to salt-induced hypertension.6 This locus, Bp1, is known to contain several genes such as the angiotensin-converting enzyme (ACE) gene and the phenylethanolamine N-methyltransferase (PNMT) gene, which may be candidates for the causal gene leading to high blood pressure. We have cloned the cDNA and genomic DNA encoding these candidates and examined differences between the strains.8 9 10 Our data so far have excluded PNMT as a candidate, but the issue of ACE is still unsettled. Our recent data from the SHRxBN rat cross have also identified five major loci linked to salt-induced hypertension.7 Other investigators have also performed linkage analysis using different rat crosses and reported several loci that cosegregate with high blood pressure, eg, the SA region on chromosome 1,11 12 the guanylyl cyclase A/atrial natriuretic peptide receptor region on chromosome 2,7 13 and the neuropeptide Y region on chromosome 4.14 It is interesting to note that several common loci that cosegregate with salt-induced hypertension can be identified among the different rat crosses6 7 11 12 13 14 15 16 17 18 (Table 1Down). These data suggest that although hypertension is a polygenic disease, there are major genes that appear to be common to salt-induced hypertension among several rat strains. However, the current genetic linkage map is not optimal for positional cloning because of the large intervals between the genetic markers. A more detailed genetic map of the rat, especially around and within the identified loci, is being produced.



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  		Figure 1. Diagram showing use of genetic models in vascular research. A, One of the most valuable contributions of inbred genetic models is for the mapping and identification of the genes responsible for disease. In this example, a spontaneously hypertensive rat (SHR) is crossed with a normotensive Wistar-Kyoto (WKY) rat. Molecular analysis of the genotype and physiological analysis of the phenotype of the animals in the F2 generation has aided in the identification of candidate genes (see Table 1Up) involved in the development of hypertension. MAP indicates mean arterial pressure. B, The use of transgenic technology has allowed the introduction of specific genes into mice or rats. In this example, the transgene is coinjected along with a gene encoding tyrosinase into a fertilized oocyte. The oocyte is implanted into a pseudopregnant female and allowed to develop. Identification of animals harboring the transgene is simplified by the presence of the tyrosinase gene, since this gene product will produce coat color in an albino animal. C, As described in detail in the text and elsewhere,54 the use of homologous recombination allows the selective disruption of a gene in mouse embryonic stem (ES) cells. After injection of these cells into blastocysts and reimplantation of the blastocysts into pseudopregnant foster mothers, chimeric animals, a portion of whose cells derived from the ES cells can be obtained. Further breeding will allow the production of animals in which all the cells contain a disruption of the gene of interest. D, Multiple techniques are available for the in vitro and in vivo transfer of genes into cells or tissues. In this example, DNA-laden liposomes are coated with UV-inactivated hemagglutinating virus of Japan (HVJ), which allows the highly efficient uptake of the DNA into cultured cells or tissues in vivo.75


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  		Table 1. Summary of the Genetic Mapping of Rat Hypertension

In addition to the mapping of genes in existing inbred strains, the construction of congenic animals will further facilitate gene identification. Congenic animals are animals of one strain into which a single locus from a second strain has been introduced by selective breeding. Congenic rats can serve two roles: physiological characterization and positional cloning. The availability of congenic animals that contain only one of the hypertensive loci/genes on an otherwise normotensive background (or conversely, a normotensive gene on a hypertensive background) can provide the opportunity to study the physiological abnormalities related to a specific locus. This approach will enable the localization of specific genetic regions and permit the dissection of the specific physiological aberrancy linked to an individual locus. Furthermore, crossbreeding the congenic strains permits analysis of gene-gene interaction, ie, additive, subtractive, and synergistic effects. Traditionally, construction of a congenic strain has required 10 generations of backcrosses and continuous selection for the phenotype. With the advent of molecular genetics and genetic linkage maps, it should be possible, by use of marker-assisted selection, to construct a congenic line in 3 to 5 generations.19 20 Indeed, Pravenec and colleagues21 presented data describing the use of congenic rats to examine the role of a chromosome 20 locus in hypertension. They demonstrated that replacement of this region of chromosome 20 of the SHR with the same region from a normotensive strain significantly lowered blood pressure in the congenic rats.

Another use of the inbred genetic models of vascular disease is for the development of novel therapies. Genetically hypertensive rats and hyperlipidemic rabbit models have long been used for testing antihypertensive and lipid-lowering drugs, respectively. Recently, the WHHL rabbit has also been used for pioneering gene therapy.22 23 24 25 26 In contrast to the hypertensive animal models, the genetic defect of the WHHL rabbit is known. As in the clinical syndrome of familial hypercholesterolemia (FH), these rabbits harbor a defect within the LDL receptor gene, resulting in no detectable LDL receptor activity in the homozygote. These rabbits exhibit high levels of cholesterol, especially LDL cholesterol. In homozygous animals, serum cholesterol ranges from 400 to 800 mg/dL, and like human homozygote FH patients, they develop vascular lesions starting at an early age. In initial gene therapy tests,23 24 liver hepatocytes were isolated from a WHHL rabbit and transfected in cell culture with an expression vector encoding the human LDL receptor. These genetically modified cells were then infused back into the liver. The retention of genetically modified hepatocytes in the liver resulted in a drop in plasma cholesterol of 200 to 400 mg/dL that persisted for up to 4 months. This ex vivo gene therapy approach is now under investigation for therapy of human homozygote FH. To eliminate the need for prior surgery of the recipient and enhance clinical feasibility, an in vivo transfection approach was recently attempted.25 Intraportal infusion of a recombinant adenovirus expressing the LDL receptor resulted in a significant reduction in serum cholesterol. However, this therapeutic response was transient (7 to 10 days). The temporary efficacy of this approach appears to result from an immune response that develops in response to transfection with the adenoviral vector. Future research will focus on developing new adenoviral vectors that maintain transfection efficiency, yet fail to activate the immune system. The WHHL rabbit is an excellent example of how an inbred genetic model has facilitated the development of gene therapy for the potential treatment of human vascular disease.


*    Transgenic Animal Models
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowInbred Genetic Animal Models...
 *Transgenic Animal Models
down arrowGene Targeting and Knockout...
 down arrowIn Vivo Gene Transfer...
 down arrowConclusions
down arrowReferences
 
The above-described genetic mapping approach of inbred animal models has yielded clues concerning the genes that may be important in the pathogenesis of vascular disease but does not provide proof for their primary causal role. Genetic analysis can only narrow the interval on the genome to 0.5 to 1.0 centimorgan, or roughly a few hundred thousand base pairs. This is potentially 50 to 100 genes! One may find certain candidate genes within this interval. However, proof of the causal relation is lacking. To fulfill Koch's postulates, one would need to substitute the suspected gene for the normal gene in an unaffected individual to see whether the disease phenotype developed. With the recent advances in recombinant technology, it is now possible to introduce specific foreign genes into fertilized oocytes, which results in the integration of the foreign DNA into the genome. Transgenic technology has produced genetically engineered animals containing an altered gene or overexpressed gene that are useful for studies of vascular disease. In simple terms, this technology (Fig 1bUp) involves the microinjection of foreign DNA into the male pronucleus of fertilized oocytes and reimplantation of the oocytes into pseudopregnant female mice. This foreign DNA is integrated into the genomic DNA of the oocyte. Offspring are screened for the presence of the transgene and bred to homozygosity, and the appropriate phenotype is examined. The technique of microinjection has become relatively easy, and large numbers of transgenic animals have been produced.

In terms of vascular disease, this technology has been used successfully in studies of hypertension as well as atherosclerosis. One of the first transgenic mice producing proteins important for blood pressure regulation was developed by researchers using transgenic technology to examine the DNA sequences that regulate the temporal and tissue-specific expression of the renin genes.27 28 29 30 It is interesting to note that none of these mice developed hypertension from expression of the transgene. In a second series of studies, Sigmund and coworkers30 31 32 33 34 produced transgenic mice carrying the renin promoter-SV40 T-antigen fusion gene that developed tumors of mesenchymal and vascular nature. We reported that the animals developed diffuse hyperplasia of arteries and arterioles in the kidney indicative of renal vascular hyperplasia.33 Importantly, renin-producing cells could be cultured from the renal tumors.34 This cell line has provided a useful in vitro system for studies of renin gene expression and prorenin processing.

Mullins et al35 developed a hypertensive rat expressing a mouse Ren-2 transgene. In this transgenic rat, the hypertension occurred despite low renal and plasma renin activities and low circulating levels of angiotensin II (Ang II). The high expression of renin in the adrenal gland suggested that aberrant expression of the renin-angiotensin system in extrarenal tissues promotes the development of hypertension in these animals and provides strong evidence for the importance of tissue angiotensin in the pathogenesis of hypertension.

Steinhelper et al36 produced transgenic mice harboring the mouse atrial natriuretic peptide (ANP) gene fused to a mouse transthyretin promoter that ensured high expression of the transgene in the liver. These animals showed a significant decrease of blood pressure associated with an eightfold elevation in circulating levels of ANP. This model will be useful to evaluate the chronic effects of high circulating levels of ANP on the cardiovascular system, which may improve our understanding of the physiological role of this peptide as well as its potential applications as a therapeutic agent.

In the area of atherosclerosis, in the 1980s, Paigen and coworkers3 4 inaugurated the study of murine atherosclerosis by identifying susceptible strains of mice, developing an atherogenic high-fat diet (1.25% cholesterol and 0.5% cholic acid), and defining a quantitative assay of lesion area in the aorta. Fatty streak lesions in the aorta are typically measured with lipid stains after about 3 months on the atherogenic diet. After longer periods, susceptible strains of mice develop more advanced lesions containing lipid-filled macrophages and smooth muscle cells, connective tissue elements, necrotic debris, and cholesterol crystals.37 Although vascular pathology in mice may be different in some respects from that in humans, the considerable power of mouse genetics and transgenic technology has made this animal model a major contributor to the study of atherosclerosis.

Paigen's initial studies established the differences in atherosclerosis susceptibility of inbred laboratory strains. Subsequently, several genetic loci defining this susceptibility were mapped in recombinant inbred strains.38 Transgenic techniques have now allowed single genes to be either overexpressed or eliminated. For complex diseases such as atherosclerosis, this allows the assessment of the action of individual molecules on disease, with the possibility of evaluating hypotheses developed by human epidemiological studies and more complex in vivo models. One of the first applications of transgenic techniques to atherosclerosis research was the overexpression of the human LDL receptor in transgenic mice, followed by the production of LDL receptor deficient mice by homologous recombination.39 40 These studies confirmed the key role of this receptor in lipoprotein metabolism and the control of hypercholesterolemia, defined the feasibility of replacing LDL receptor function by virus-mediated gene delivery, and allowed in vivo determination of structure-function relations.40

The gene for human apolipoprotein(a) has been introduced into mice, resulting in susceptibility to atherosclerosis in a normally resistant genetic background.41 42 Apolipoprotein(a) is the distinguishing protein component of the lipoprotein Lp(a), which otherwise resembles LDL. Elevated blood concentration of elevated Lp(a) is an independent, inherited risk factor for myocardial infarction, stroke, and restenosis (see References 43 and 44 for review). The apolipoprotein(a) gene is a close homologue of plasminogen that may have arisen by duplication of the plasminogen gene during primate evolution.45 Apolipoprotein(a) is absent from rodents and perhaps all nonprimate species that have been used as models of human vascular disease. Transgenic mice expressing human apolipoprotein(a) developed a 20 times greater area of lipid-rich lesions than control mice after 3 months on the atherogenic diet.42 This observation directly confirmed the atherogenicity of apolipoprotein(a) and established a model that could be manipulated chemically, physically, or by further genetic means to elucidate the activities of apolipoprotein(a) and help establish modes of controlling its deleterious effects. For example, in these transgenic mice, human apolipoprotein(a) associates very poorly with mouse LDL compared with human LDL.41 This suggests that even free apolipoprotein(a) not bound to a lipoprotein particle could exert atherogenic actions. One of these is the reduction of fibrinolysis by apolipoprotein(a) as a result of its inhibition of its homologue, plasminogen. Although documented in several in vitro studies, this hypothesis has been difficult to support by correlating Lp(a) levels and clot lysis time in human blood samples (see Reference 46 for review). This could be a result of the inability of whole blood measurements to reflect local clot lysis or of the difficulty in accounting for other variables in human population studies. It has recently been possible to prepare radioactive human blood clots and inject them into the mouse circulation, where their lysis can be followed by external detection. With these techniques, it has been shown that apolipoprotein(a) retards clot lysis in vivo (T. Palabrica, B. Furie, personal communication). In another application of these transgenic mice, Grainger and colleagues47 extended in vivo their observations based on cell culture48 that apolipoprotein(a) inhibits the plasmin-dependent activation of transforming growth factor-ß (TGF-ß), which in turn hinders the suppressive effects of TGF-ß on smooth muscle cell proliferation48 and migration.49 Removal of mouse vessels for quantitative fluorescence staining shows that focal accumulation of apolipoprotein(a) in lesion-prone areas colocalizes with significant reduction in plasminogen and latent TGF-ß activation, and alteration in smooth muscle cell phenotype is revealed by osteopontin staining.47

As will be discussed in more detail in the next section, multiple recombinant strains of mouse have been produced in which the expression of various genes involved in human lipid transport and metabolism has been altered. They have proved to be extremely useful in defining the roles of a number of gene products in the complex pathways in lipid transport and metabolism (for review see Reference 50). Transgenic techniques have also been used to study the control of expression of apolipoprotein genes. In particular, such studies were essential in identifying the remote element that establishes liver-specific expression of the linked apolipoprotein E and C-I genes and the separate liver and intestine expression elements of the linked apolipoprotein A-I, C-III, and A-IV genes.51 52 A particularly exciting recent development is the production of transgenic rabbits that overexpress human apolipoprotein E and hepatic triglyceride lipase. (Fan et al. Proc Natl Acad Sci U S A. 1994;91:8724-8728.) Since rabbits offer several advantages over mice, including larger vessel size and greater sensitivity to moderate-fat diets, one expects to see further use of transgenic rabbit technology in the study of cardiovascular disease.


*    Gene Targeting and Knockout (Homologous Recombination)
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowInbred Genetic Animal Models...
 up arrowTransgenic Animal Models
*Gene Targeting and Knockout...
down arrowIn Vivo Gene Transfer...
 down arrowConclusions
down arrowReferences
 
Transgenic animals obtained by microinjection of foreign DNA are useful as genetic models of vascular disease. However, this approach has limitations as a method to examine the role of an endogenous gene in development and disease pathophysiology. They do not truly fulfill Koch's postulates, since the foreign gene is expressed in the presence of the wild-type gene and is integrated at random into the genome. Gene targeting with replacement of the wild-type gene by the foreign gene is a more ideal approach.

The technique of homologous recombination in murine embryonic stem cells has made it possible to selectively disrupt or mutate genes in mice. This is a potentially powerful tool for examining the role of genes in cardiovascular physiology and development. The process of generating these genetically altered strains of mice is outlined in Fig 1cUp and has been more extensively reviewed elsewhere.53 Preimplantation blastocysts are harvested from 129sv mice and disaggregated to establish embryonic stem cell lines. These cells are grown under special conditions to preserve their undifferentiated state. While in culture, these cells can be transfected with the targeting vector, a fragment of the gene of interest into which a selectable marker (neomycin resistance) has been inserted to functionally disrupt the gene. In a small number of cells, the modified gene is exchanged for the endogenous gene by homologous recombination. Cells having this gene disruption will also express neomycin, which is useful for isolating pure clones of cells having the targeted disruption. These genetically modified ES cells are then injected into blastocysts harvested from C57 mice (which have a different coat color marker than 129 mice). The injected blastocysts are implanted into pseudopregnant CD1 mice, where both C57 cells and embryonic stem (ES) cells compete for the formation of various tissues in the embryo. If the chimeric mice that develop from these implanted blastocysts have germ cells derived from the ES cells, the gene modification can be passed on to their offspring.

To date, the most atherogenic mouse strain produced resulted from knockout of the apolipoprotein E gene.54 55 Apolipoprotein E plays important roles both in the catabolism of highly atherogenic remnant lipoprotein particles and in reverse cholesterol transport. Apolipoprotein E knockout mice achieve circulating cholesterol levels of {approx}500 mg/dL on low-fat and nearly 2000 mg/dL on high-fat diets and evidence massive lesions on both diets. Analysis of the advanced lesions in these mice has revealed that mice can develop vessel pathology with significant similarity to that seen in advanced human atherosclerosis and are proving to be valuable models of murine vessel pathophysiology.56 57 Nearly 20 genes involved in human lipid transport and metabolism have now been either overexpressed or knocked out in transgenic mice (see Reference 50 for review). These include most of the apolipoproteins associated with HDL structure and metabolism, including apolipoproteins AI and AII, cholesteryl ester transfer protein, and hepatic triglyceride lipase. Some of the results of studies with these mice include demonstration of the cardioprotective role of apolipoprotein AI- but not AII-containing HDL subspecies and the role of cholesteryl ester transfer protein in creating a more "humanlike" lipid density profile, with a concomitant increase in the development of atherosclerosis.58 59 60 61 62 Interestingly, crossbreeding of transgenic mouse strains that overproduce apolipoprotein AI-containing HDL alleviates the atherogenicity of either apolipoprotein(a) overexpression or apolipoprotein E knockout, each of which presumably causes atherosclerosis by quite different mechanisms.63 64 This affirms the protective nature of high HDL concentration revealed by epidemiological studies in humans and provides a manipulatable in vivo model with which to investigate the role of HDL in reverse cholesterol transport and protection against oxidation of lipoproteins.

Gene disruption studies are particularly useful for identifying the role of specific genes in development. Although this technique is relatively new and its application to cardiovascular medicine has been limited, it has provided insight into the role of several transcription factors in muscle development. Both MyoD and Myf-5 have been implicated in muscle development and are capable of inducing muscle differentiation when ectopically expressed in nonmuscle cells.65 It was therefore surprising that the disruption of the MyoD gene66 or the Myf-5 gene67 in mice did not interfere with muscle development. Of interest, however, Myf-5 expression was increased 3.5-fold in mice lacking a functional MyoD gene. In mice lacking functional genes for both Myf-5 and MyoD, there is a complete disruption in skeletal muscle development.68 These studies therefore provide evidence for redundancy in transcription factors that regulate muscle development. They also demonstrate that transcript factors can have multiple roles in development. Mice lacking Myf-5 expression exhibited an unexpected phenotype, the failure of rib development, demonstrating an unexpected role for Myf-5 in development. In contrast to the results of MyoD and Myf-5 gene disruption, the disruption of a single gene for another muscle-specific transcription factor, myogenin, led to a severe defect in skeletal muscle development.69

We have recently succeeded in disrupting the genes encoding various adrenergic receptors as well as the genes encoding isoforms of the angiotensin receptor (unpublished data). The detailed examination of these animals should yield information on the role of multiple receptor isoforms on cardiovascular growth and development as well as cardiovascular physiology.

The fact that genes involved in cardiovascular function or development may have multiple functions can complicate the evaluation of gene disruption experiments. For example, the disruption of the gene for the vasoactive peptide endothelin led to lethal abnormalities in craniofacial development in animals homozygous for the disruption,70 precluding analysis of cardiovascular physiology in these mice. Nevertheless, it was possible to derive some physiological information from animals heterozygous for the endothelin gene disruption. These animals had normal craniofacial development and were viable and fertile but exhibited a higher blood pressure than control littermates. This is surprising, since endothelin is a potent vasoconstrictor and one would have predicted that the reduced circulating endothelin levels in the heterozygous animal would have been associated with a lower systemic blood pressure. These results demonstrate a more complex role for endothelin in regulating hemodynamics.

The utility of this approach to studying cardiovascular physiology in adult animals may be limited when developmental abnormalities are severe. However, recent studies suggest that it may be possible to overcome this limitation. Gu and coworkers recently described an approach to limit gene disruption to a specific cell type.71 This approach involves using the techniques outlined above to introduce two short nucleotide sequences called loxP sites on either side of a critical region within the gene of interest. The insertion of these sites will not disrupt gene function unless the cell also expresses Cre recombinase (derived from bacteriophage P1). Cre recombinase recognizes the two loxP sites and removes all of the DNA sequences between these sites. If animals carrying the loxP modified gene are mated to animals carrying a transgene with Cre recombinase expressed under the control of a tissue-specific promoter, a portion of the offspring will inherit both the loxP modified endogenous gene and the Cre recombinase transgene. In these mice, the expression of Cre recombinase in the specific tissue will lead to the disruption of the loxP modified gene only in that tissue. Such creative application of recombinant DNA techniques will further enhance the utility of in vivo modification in the future.

Perhaps the principal disadvantage of the use of in vivo gene modification is that it currently can be done only in mice, which have not been used extensively for cardiovascular research. In the future, it may be possible to adapt these techniques to other species. For example, the establishment of a rat ES cell line that can produce chimeras has just been reported by Lannaccone et al,72 suggesting that homologous recombination may soon be possible in the rat. However, because time and animal costs will always be important considerations, it is likely that most in vivo gene modification studies will be done in mice. Therefore, it is essential for molecular biologists and cardiovascular physiologists to join forces to better characterize cardiovascular physiology and development in mice.


*    In Vivo Gene Transfer Approach
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowInbred Genetic Animal Models...
 up arrowTransgenic Animal Models
up arrowGene Targeting and Knockout...
 *In Vivo Gene Transfer...
down arrowConclusions
down arrowReferences
 
The effect of gene manipulation is usually seen throughout development. Hence, these techniques as currently practiced may not be as useful as desired to study the roles of specific molecules in the pathophysiology of adult vascular disease. In vivo transfer technology provides the capability of examining adult vascular pathobiology. For example, many substances that are synthesized in the cardiovascular system have been postulated to exert autocrine-paracrine actions on vascular function and/or structure. These include growth factors, cytokines, adhesion molecules, and vasoactive substances. However, definitive proof for their functions cannot be obtained because in vivo studies are limited by the multiplicity of coexisting variables, the difficulties in manipulating individual components, and the methodological limitations in studying the function of locally produced modulators in the absence of any contribution by the circulation. Cell culture and gene transfer technologies have provided us with the opportunity to study cellular responses to the manipulation of the individual components of a putative autocrine-paracrine mediator system (ie, by overexpression or inhibition). An in vivo experimental approach that can selectively manipulate vascular wall gene expression would be useful for furthering our understanding of the biology and pathobiology of autocrine-paracrine systems.

Recent progress in in vivo gene transfer technology provides us with this possibility. The feasibility of transfecting blood vessels with foreign DNAs in vivo was described previously by use of retroviral vectors.73 74 75 As an alternative, direct physical transfer of genes into intact blood vessels in vivo has been reported by use of cationic liposomes (Lipofectin) as the carrier of DNA.75 Recently, the adenoviral vector transfer method has also been used successfully for gene transfer into the blood vessel.76 77 Several functional genes have been successfully transferred into endothelial and vascular smooth muscle cells (VSMCs) in vivo. For example, transfection of the platelet-derived growth factor gene induced VSMC proliferation,78 whereas transfection of the fibroblast growth factor gene caused angiogenesis in association with VSMC accumulation.79 These results emphasize the potential utility of in vivo gene transfer technology to define the biological role of locally generated vasomodulators within the vessel wall in vivo.

To further define the biological role of locally generated vasoactive factors, we recently used hemagglutinating virus of Japan (HVJ)–liposome-mediated gene transfer (Fig 1dUp; Reference 80) into the vascular wall.81 This method yields high transfection efficiency (up to 30% of vessel wall cells may be transfected) without apparent toxicity. The usefulness of HVJ-liposome gene transfer for in vivo vascular biology studies has permitted us to examine the role of autocrine-paracrine systems in the vessel wall. The role of vascular ACE as a determinant of vessel tone and structure is a controversial subject in hypertension research. Given the difficulties of distinguishing between circulating versus vascular ACE effects, traditional experimental approaches using pharmacological tools have provided only indirect evidence of the potential importance of vascular ACE as a determinant of local Ang II generation. Using the in vivo gene transfer, we tested the hypothesis that increased local expression of vascular ACE and production of Ang II are important determinants of vascular structure. Our data demonstrate that increased local expression of ACE within vessel wall promotes autocrine-paracrine Ang II–mediated vascular hypertrophy in vivo.82 Thus, in this study, the use of in vivo gene transfer has enabled us to address two important questions in hypertension and vascular research: (1) Can angiotensin directly mediate vascular hypertrophy independent of its blood pressure effect? (2) Is local tissue ACE important in regulating local vascular function and in contributing to pathophysiology?

Endothelium-derived nitric oxide may be an important endogenous inhibitor of vascular lesion formation. In addition to its vasorelaxant effect, nitric oxide has been shown to inhibit VSMC proliferation in vitro. Furthermore, it inhibits platelet aggregation and leukocyte adhesion. However, these data are derived from in vitro studies. There is no direct evidence that endogenously generated nitric oxide is vasculoprotective in the intact animal. We tested whether the restoration of nitric oxide production by in vivo gene transfer of the gene encoding endothelial cell–type nitric oxide synthase (ec-NOS) could inhibit neointima formation after balloon injury. To assess the effect of the biological response to transfection of the ec-NOS gene on vascular lesion formation in vivo, we chose the well-characterized rat carotid artery balloon injury. Using the HVJ–liposome-mediated gene transfer approach, we achieved high expression levels of ec-NOS in injured rat carotid arteries, restored local nitric oxide generation, and demonstrated a marked reduction (70%) in neointimal hyperplasia in ec-NOS-transfected vessels compared with either control vector-transfected vessels or untreated injured vessels.83 These results demonstrate that in vivo gene transfer of the ec-NOS gene inhibits neointima hyperplasia via the increased local generation of nitric oxide within the vessel wall. This study provides direct evidence that the regulation of local nitric oxide generation is an important determinant of vascular lesion formation in vivo.

Another application of in vivo gene transfer is to study the unknown function of a novel molecule using a gain-of-function approach. For example, although the type 1 Ang II (AT1) receptor is well characterized, the function of the type 2 (AT2) receptor remains an enigma. With a gain-of-function approach using in vivo gene transfer, we tested the hypothesis that the AT2 receptor can modulate the growth of VSMCs. We transfected an AT2 receptor expression vector into balloon-injured rat carotid artery—a model of in vivo smooth muscle migration and proliferation—and observed that overexpression of the AT2 receptor decreased in vivo vessel wall DNA synthesis and attenuated neointima formation.84 Furthermore, in cultured VSMCs, we demonstrated that AT2 receptor transfection resulted in reduced rates of cell proliferation that is stimulated by AT1 receptor activation. These results suggest that the AT2 receptor exerts an antiproliferative effect on cell growth and counteracts the growth action of AT1 receptor. These results have important implications in our understanding of the role of locally generated vasoactive substances in the process of vascular remodeling.

Advances in molecular and cellular biology have begun to identify critical mediators that may be involved in the processes of atherogenesis, plaque rupture, thrombosis, ischemia, cell death, and the healing response after infarction. Moreover, the capacity to manipulate the gene expression of these putative mediators will enable investigators to begin to define the role of these factors as determinants of myocardial infarction and its sequelae. It is hoped that these studies will also provide new directions for innovative therapies to prevent plaque rupture or promote adaptive ventricular remodeling after infarction. For example, "proof-of-concept" studies already performed document that gene transfer techniques can be used to express genes that promote angiogenesis and collateral formation with the intent of modifying the ventricular structure after infarction or preventing recurrent infarction.85 Thus, advances in in vivo genetic engineering technologies to modify gene expression within the vasculature may have a profound impact on our understanding of myocardial diseases.

Thus, in vivo gene transfer is suitable to study vascular biology by local overexpression or inhibition of specific genes. In vivo gene transfer technology has the following advantages over the conventional experimental approaches such as classical pharmacology: (1) the target gene can be transfected into a local segment of a blood vessel or myocardium, thereby avoiding a systemic effect; (2) this transfected segment can be compared with adjacent untransfected segments or with the contralateral control blood vessel, which are subject to the same hemodynamics and circulating humoral factors; and (3) the consequences of local overexpression within the physiological/pathophysiological range of the target gene may be studied. We anticipate that future studies will define the pathobiological role of autocrine-paracrine systems in the local regulation of cardiovascular function and structure.

Local gene transfer technology also provides us the opportunity to treat cardiovascular diseases. In several cardiovascular diseases, eg, restenosis after angioplasty and accelerated transplant coronary vasculopathy, no known effective therapy exists. Somatic gene therapy is the introduction of normal genes into the somatic cells of patients to correct an inherited or acquired disorder through the synthesis of specific gene products in vivo. In general, there are three methods of gene modification: (1) gene replacement, (2) gene correction, and (3) gene augmentation. Although current in vivo methods for vascular gene transfer are still limited by lack of efficiency and potential toxicity,75 future advances may provide the opportunity to treat vascular diseases such as hypertension by manipulating vasodilatory and growth-inhibitory genes such as ANP or nitric oxide synthase.

Another attractive strategy involves an anti-gene approach in which targeted genes are inactivated by antisense oligonucleotides or plasmids. Such an approach can define the contribution of various factors to vascular pathobiology and may be used for therapy. To define the feasibility of an anti-gene approach to modulate vascular structure in vivo, our initial studies have examined the determinants of VSMC growth in vivo in the rat carotid balloon-injury model. Neointimal formation after balloon injury involves a complex interaction between multiple growth factors that promote VSMC proliferation and migration, making it unlikely that selective inhibition of a particular growth factor will completely prevent lesion formation. However, growth factor–induced cell proliferation involves the sequential activation of intracellular proteins that promote cell cycle progression. Accordingly, we hypothesized that VSMC proliferation and lesion formation could be prevented by the blockade of genes regulating cell cycle progression—the final common pathway. Indeed, we recently showed that intraluminal in vivo gene transfer of antisense oligonucleotides against the cell cycle genes proliferating cell nuclear antigen and cdc 2 kinase markedly inhibited VSMC proliferation and lesion formation after injury in vivo.86 Similarly, Simons et al87 reported the inhibition of neointimal formation by periadventitial administration of antisense oligonucleotides against c-myb.

Recently, Ohno et al88 demonstrated that adenovirus-mediated gene transfer of the herpes virus thymidine kinase gene into injured porcine arteries, accompanied by administration of ganciclovir, resulted in destruction of proliferating VSMCs and consequently in inhibition of neointimal hyperplasia. We speculate that continued development of these methodologies will facilitate the use of oligonucleotide and gene transfer technology to further characterize the biological role of genes activated during the pathogenesis of specific cardiovascular diseases as well as provide potential new therapeutic agents for use in humans.


*    Conclusions
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowInbred Genetic Animal Models...
 up arrowTransgenic Animal Models
up arrowGene Targeting and Knockout...
 up arrowIn Vivo Gene Transfer...
 *Conclusions
down arrowReferences
 
In this article, we present the major technologies in use for developing new genetic models for vascular research. Obviously, no one technique is suitable for all scientific inquiries, and each technique has its own strengths and weaknesses (Table 2Down). Transgenic technology is a powerful tool to examine the effects of overexpression or ectopic expression of a gene product. However, the technology is hindered by several drawbacks. Transgenic techniques are well suited for the examination of the effects of overexpression of circulating proteins, as is the case with the apolipoproteins. At this time, the examination of the local autocrine or paracrine effects of the expression of a substance, independent of the systemic effects, can be difficult. However, it must be remembered that the establishment of tissue-specific and regulatable promoters is a major goal of transgenic research. Another drawback to transgenic techniques is that these techniques are costly and time-consuming.


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  		Table 2. Comparison of In Vivo Gene Manipulation Methodologies

Homologous recombination is also a powerful technique that can provide information that cannot be obtained by other technologies. For example, few if any techniques are currently available to examine the effects of blockade of expression of a protein during embryonic and fetal development. However, potential embryonic lethality of the knockout will severely affect the usefulness of the model. Currently, knockout technology suffers from many of the same drawbacks of transgenic techniques, such as the inability to disrupt a gene in a temporal or tissue-specific manner. Promising new techniques such as the use of Cre recombinase may circumvent these technical limitations.71 89 Like transgenics, homologous recombination can be costly and time-consuming.

In vivo transfer is relatively simple and inexpensive compared with transgenic and knockout methods. The ability to express or block the expression of a protein in a temporal and tissue-specific manner by use of in vivo gene transfer is another advantage, as is the ability to examine the local effects of a protein independent of systemic effects. However, this technology also has several drawbacks. The effects of in vivo gene transfer are usually short lived. The technique is useful for studying local effects but not as effective for studying simultaneous expression of a gene in multiple tissues. Although this technique is ideally suited to acute situations, its use in chronic studies is limited.

In summary, novel techniques are now available for studies of vascular biology/disease and for the generation of new genetic models of vascular disease. It is becoming increasingly clear that we are on the threshold of an exciting period of scientific investigation in which the technology of genetic manipulation and engineering is being used as an experimental tool to define the pathobiology of cardiovascular disease. The capacity of these technologies to inhibit or augment gene expression in vivo will enable investigators to establish a more powerful link between the disciplines of physiology, pharmacology, cell biology, molecular genetics, and cardiovascular medicine and to develop novel therapies.


*    Acknowledgments
 
This work was supported by National Institutes of Health grants HL-46631, HL-35252, HL-35610, HL-48638, and HL-07708 and by the American Heart Association–Bugher Foundation Center for Molecular Biology in the Cardiovascular System. We are grateful to the AHA–Bugher Foundation, whose support has successfully fostered training and research in these areas at Stanford.

Received August 29, 1994; accepted October 24, 1994.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowInbred Genetic Animal Models...
 up arrowTransgenic Animal Models
up arrowGene Targeting and Knockout...
 up arrowIn Vivo Gene Transfer...
 up arrowConclusions
*References
 

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