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a Department of Urology, College of Physicians and Surgeons of Columbia University, New York, New York 10032
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Penile tissue was obtained from adult rats and from human patients undergoing penile prosthesis implantation. Analysis of the forms of VEGF transcripts was performed using a reverse transcription-polymerase chain reaction technique with primer pairs derived from the first and eighth exon of the VEGF gene. The expression levels of the various isoforms in the rat penis were then quantified using RNase protection assays. Four previously described splice variants of VEGF mRNA (VEGF 120, 144, 164, 188) were detected in rat and human penile tissues. In contrast to what is seen in the rat lung, where the most abundant form of VEGF mRNA is the 188 splice isoform, VEGF 164 is the most abundant transcript detected in the penis. Finally, sequence analysis of numerous VEGF cDNA clones obtained from the rat penis demonstrated the presence of a previously undescribed VEGF splice variant that could give rise to a protein of 110 amino acid residues (VEGF 110, GenBank accession no. AF080594). In summary, a number of VEGF mRNA isoforms are expressed in the rat and human penis, with the splice variant encoding a 164-amino acid protein present in greatest abundance. This study is a prelude to attempts to genetically manipulate VEGF expression in the penis as a therapy for erectile dysfunction.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Erection is a hemodynamic phenomenon involving the tissue of the corpora cavernosa as well as the corpus spongiosum in the penis. This tissue is a complex admixture of smooth muscle, endothelial cells, fibroblasts, and nerves interacting under stimulatory conditions to drastically enhance and maintain an accessory blood supply that imparts rigidity to the penis. Given the need for stringent control of blood flow during this response, it is no surprise that vascular insufficiency has the ability to drastically suppress erectile capability. In fact, penile vascular insufficiency is believed to be a very common pathomechanism of erectile dysfunction [2]. This is associated with substantial pathologic changes in the erectile tissue leading to reduction in vascular smooth muscle cells and increases in collagen and fibrosis [3–6].
The currently available treatments of erectile dysfunction induce temporary erections at the time of administration of such treatments. However, these treatments do not address or cure the basic vascular/cavernosal pathology causing erectile dysfunction. The rationale for a curative treatment that improves or repairs the vascular structure and function of the erectile tissue is based upon an understanding of erectile physiology and the pathophysiology of vascular erectile dysfunction. Consequently, it is prudent to pursue investigation of factors that induce new vascular structure formation or vasculogenesis. Several naturally occurring growth factors have been found to induce vasculogenesis [7–10]. Such growth factors include the vascular endothelial growth factor (VEGF) family [11], the fibroblast growth factor (FGF) family [12, 13], transforming growth factor 
and ß [14], and platelet-derived growth factor [15–17]. VEGF may well be one of the most potent and interesting of these vascular growth factors. It is thought to play a role in embryonic vasculogenesis [18], maintenance of vascular structures in the adult, and formation of new blood vessels in the adult in response to ischemia and other pathologic states. Therapies that increase tissue levels of VEGF in laboratory animals or humans with peripheral vascular disease have resulted in a measurable increase in tissue vascularity [19–24].
In order to contemplate the potential of VEGF therapy for erectile dysfunction, we first wanted to characterize the extent to which VEGF is expressed in the mammalian penis and in erectile tissue, as well as to determine which of the known isoforms of VEGF (derived from mRNA splice site variation) are most abundantly present in this tissue as compared to other mammalian tissues. Here we present our preliminary survey of VEGF isoform expression (mRNA) in the mature rat penis and in human erectile tissue.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Specimens of cavernosal tissue of 12 humans (ages 39–79) were surgically removed from the penoscrotal junction at the time of penile prosthesis implantation under an Institutional Review Board-approved protocol. These biopsies measured approximately 5 x 5 x 10 mm. The specimens were immediately frozen under liquid nitrogen and were stored at -70°C until processing.
Laboratory Animals and Tissues
Age-matched male Sprague-Dawley rats (8 wk; Camm Laboratories, Camden, NJ) were obtained under a protocol approved by the Institutional Animal Care and Use Committee and were maintained on a 12-h light:dark cycle with food and water available ad libitum. Rats were killed by a lethal overdose of sodium pentobarbital. Lung and penis were harvested immediately. Penile tissue was obtained through a circular incision made at the corona with subsequent removal of the foreskin and the shaft skin and amputation of the penis. The glans, which contains a significant amount of skin, was also removed. Excess blood was removed by gently blotting the penis on sterile cotton gauze, and the specimen was flash frozen in liquid nitrogen and stored at -70°C until use. This procedure provided cryopreserved, de-skinned penile shafts that contained mostly erectile tissue.
RNA Extraction
Tissue (pooled penis from 20 rats or pooled specimens from 12 humans) was first pulverized under liquid nitrogen to a fine powder. Total RNA was isolated from the tissue powder using an RNAzol (Tel-Test, Inc., Friendswoods, TX) extraction procedure according to the manufacturer's protocol [25, 26]. RNA concentration was determined by spectrophotometry at 260 nm; and to ensure that the RNA was not degraded, each sample was analyzed by electrophoresis on a 1% denaturing formaldehyde gel. Total RNA was extracted from the prostate carcinoma cell line, LNCaP, using a modification of a procedure previously described [27].
Reverse Transcription-Polymerase Chain Reaction (RT-PCR) to Amplify VEGF cDNAs
One microgram total RNA was used for first-strand cDNA synthesis. The RNA was annealed to 0.5 µg oligo(dT) primer (Gibco BRL, Grand Island, NY) in a volume of 20 µl at 70°C for 10 min and then chilled on ice quickly for 2 min. Ten millimolar dNTPs, 5-strength first-strand buffer, 0.1 M dithiothreitol, 10 U RNase inhibitor (Gibco BRL), and 200 U Superscript II reverse transcriptase (Gibco BRL) in a final volume of 50 µl were added. The reaction mix was incubated by heating 37°C for 90 min, and the cDNA was stored at -20°C until used.
Polymerase chain reaction (PCR) techniques were then used to identify the presence of VEGF isoforms in the RNA samples. Primers used for the amplification assay (Table 1) were slight modifications of those published previously [28]. Because these primers initiate within the first exon and terminate within the eighth exon of VEGF-A, they enable amplification of all the known VEGF splice variants. The oligonucleotide sequences of the primers used are shown in Table 1.
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The PCR reaction mix contained 10-strength reaction buffer (100 mM Tris, 500 mM KCl, 1.5 mM MgCl2, pH 8.3), dNTP mix (10 mM each dATP, dGTP, dCTP, and dTTP at neutral pH), 100 pmol upstream primer (5'-human/5'-rat), 100 pmol downstream primer (3'-human/3'-rat), 2.5 U Taq DNA polymerase (Boehringer Mannheim Biochemicals, Indianapolis, IN), 10 µl cDNA, and sterile water to 50 µl. PCR was carried out using a DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk, CT) as follows: 94°C for 7 min; then 35 cycles of denaturation at 94°C for 1 min, annealing and extension at 72°C for 1 min each. The PCR products (20 µl of lung-rat/LNCaP and 35 µl of penis-rat/penis-human reaction mix) were analyzed by electrophoresis on a 2% agarose gel that was subsequently stained with ethidium bromide for visualization of DNA bands. A 100-base pair (bp) ladder DNA molecular weight marker was used (Boehringer Mannheim Biochemicals) to provide a size reference for the test reactions.
Cloning and Sequencing of RT-PCR Products
One microliter of penis-rat PCR products was ligated into the pGEM-T easy Vector DNA and used to transform DH5--competent cells. Transformants containing cDNA inserts were characterized for insert size by analysis of EcoRI-digested minipreps. A variety of plasmid vectors containing cDNA inserts of different sizes were then sequenced from the double-stranded templates by standard dideoxynucleotide sequencing techniques. These sequences were compared to the human or rat VEGF cDNA sequences present in GenBank to identify exon structure.
RNase Protection Assay for VEGF mRNA Isoforms
Two antisense RNA probes for VEGF cDNA were used. One was in vitro transcribed from an ApaI-digested vector containing the rat VEGF 164 splice isoform cDNA, whereas the other was in vitro transcribed from an ApaI-digested vector containing the rat VEGF 188 splice isoform DNA using SP6 polymerase. The in vitro transcription reactions were carried out in the presence of 250 µCi [32P]UTP (NEN Life Science Products, Boston, MA) using the reagents provided in an in vitro transcription kit (MAXIscript; Ambion, Inc., Austin, TX) according to the manufacturer's instructions. Individual labeled antisense probes (619 bases for the VEGF 164 probe and 691 bases for the VEGF 188 probe) were purified by electrophoresis on a denaturing 5% acrylamide gel and were then eluted from the gel into elution buffer (from RPA II kit; Ambion). These probes were used in solution hybridization procedures with 20 µg of rat lung RNA or 30 µg of penis RNA using the manufacturer's recommendations provided in the RNase assay kit (RPA II; Ambion) overnight. Hybridized specimens were digested with a nuclease cocktail provided in the RPA II kit, and the digests were subsequently analyzed by electrophoresis on 5% acrylamide sequencing gels adjacent to molecular weight marker lanes.
Some control reactions included labeled probes hybridized with yeast RNA (20 µg) (with/without RNase digestion). Gels were exposed to Kodak (Eastman Kodak, Rochester, NY) XAR-5 film to produce an autoradiograph showing the presence of RNase-protected bands.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mammalian VEGF gene [29] is known to consist of at least 8 exons that can be assembled, through alternative splicing [30–32], into a number of variant mRNA molecules that have the potential to encode differing proteins, each having mitogenic activity for the endothelial cell [33, 34]. Both human and rodent cell lines and tissues have been characterized for the expression of these VEGF splice variants, and the prominent forms found in adult tissues include transcripts that can encode for 121, 165, or 189 amino acid (AA)-containing proteins (in humans) [35–38]. Other VEGF splice variants have been detected in much lower abundance: for example, a 145 AA-encoding transcript for VEGF found in human uterine and placental tissue [39–41] and a 206 AA-encoding transcript found in fetal liver and placenta [41, 42]. Of this group of VEGF splice variants, all transcripts contain the first 5 VEGF exons as well as the last (eighth) exon, but they differ in the inclusion and/or arrangement of the sixth and seventh exons. Virtually identical VEGF splice variants have also been described in rodent tissues; however, the variant rodent VEGF transcripts generally encode for a protein product containing one amino acid less than the human counterpart (see Fig. 1) [28, 43].
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Using a RT-PCR screening method, we attempted to identify the VEGF mRNA splice variants expressed in rat and human penile tissues. Oligonucleotide primers designed to amplify cDNA from the first to the eighth exon of rat VEGF were used in a PCR amplification reaction of cDNA made from RNA extracted from rat lung or from rat penis. The RT-PCR amplification products were examined by electrophoresis on an agarose gel that was subsequently stained with ethidium bromide. As expected (Fig. 2A), these primers amplified three distinct cDNA fragments of 564, 492, and 360 bp from the rat lung cDNA (corresponding to the expected 188, 164, and 120 rat VEGF AA-encoding isoforms) as well as a distinct cDNA fragment of 432 bp. All of these cDNA fragments were also amplified from the cDNA of rat penis. Although the RT-PCR screening method was not designed to be quantitative, there appeared to be a distinct difference in the abundance of these RT-PCR products between rat lung and penis tissue. The most predominant of the VEGF cDNAs amplified from lung RNA encoded the 188-AA isoform of VEGF, whereas the most predominant transcript amplified from penis RNA appeared to be the one encoding the 164-AA isoform of VEGF (Fig. 2A). Each of these cDNA fragments was individually cloned into plasmids so that they could be sequenced and confirmed as VEGF splice variants. The sequencing confirmed our identification of the 188-, 164-, and 120-AA forms; it also confirmed that the 432-bp amplification product was the VEGF transcript variant encoding the 144-AA form of VEGF that had been previously found only in uterine and placental tissues. More surprisingly, in our survey of the cDNA clones resulting from RT-PCR amplification, we found one cDNA clone (330 bp) containing a novel splice variant of VEGF-A that has never before been described (see Fig. 1). This splice variant contained the entire first 3 exons of rat VEGF-A spliced to a partial exon 7 (now referred to as exon 7B) and was terminated by the entire exon 8. It is likely that the partial exon 7 included in this novel transcript (7B) was another of the many splice adaptions of the VEGF-A transcript, since a canonical splice donor sequence (CCGCAG/) [44] was present at the end of the region of exon 7A that was deleted from this particular splice variant. Although this splice variant must be present in much lower abundance than any of the better-known forms, this novel VEGF-A splice variant does have the potential to encode a 110-AA isoform of VEGF-A.
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Similar primer sets were utilized to amplify cDNA that was reverse transcribed from human penis RNA and from RNA extracted from a human prostate cancer cell line, LNCaP, known to express VEGF-A in abundance. Figure 2B shows the ethidium bromide-stained agarose gel on which the amplification products from human penis cDNA, LNCaP cell cDNA, or rat tissue (penis and lung) cDNA were electrophoresed. As can be seen from the results shown, the human penis (and human LNCaP cells) expresses the identical repertoire of VEGF splice variant isoforms found in the rat penis (including the 189, 165, 145, and 121 AA-encoding isoforms). Moreover, these isoforms and the rat penis isoforms were present in similar abundance.
RNase Protection Assay to Confirm Relative Abundance of VEGF-A Transcripts in Rat Penis
In order to confirm and better quantify that the predominant VEGF-A transcript splice variants expressed in the rat penis differed significantly in abundance from the forms expressed in the rat lung, we developed an RNase protection assay that would allow us to distinguish the relative abundance of the different rat VEGF-A splice variants in RNA samples. 32P-Radiolabeled probes for the 188-AA or the 164-AA splice variant of rat VEGF-A cDNA were prepared by in vitro transcription of expression plasmids containing these respective cDNA inserts. The probes were individually hybridized to RNAs extracted from rat lung or rat penis and were then digested with a nuclease cocktail and electrophoresed on a polyacrylamide gel. The gel was exposed to film for autoradiography, and the signal intensity of the protected fragments on this assay demonstrated the relative abundance of the corresponding transcript.
This two-probe assay was designed to demonstrate the presence of the VEGF isoforms through the detection of certain "diagnostic" fragments. For example, with use of the larger transcript as a probe (the 188 splice isoform), the presence of a protected fragment at 564 bp would distinguish and confirm the presence of the 188 splice variant in the test mRNA specimen, whereas the presence of a protected 150-bp fragment in the digest would distinguish the presence of the 164 splice variant (see diagram at lower part of Fig. 3A). Likewise, with use of the smaller transcript as a probe (164 splice isoform), the presence of a protected fragment of 492 bp would confirm the presence of the 164 splice variant in the test specimen (see diagram at lower part of Fig. 3B).
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Our results (Fig. 3, A and B) for this RNase protection assay showed that in the rat lung, the most abundant splice variants of VEGF-A mRNA were for the 188 and the 164 isoforms, confirming the earlier experiment involving RT-PCR amplification of VEGF-A isoforms (Fig. 2, A and B) as well as other reports [39]. In rat penis mRNA, we did not observe significant protection of a 564-bp fragment or the 414-bp fragment using the larger splice isoform probe, indicating a much lower abundance of the 188 splice variant mRNA in penis mRNA as compared to lung mRNA, as well as a low abundance of the 144 splice variant in penis mRNA. With the smaller transcript as a probe, we were able to identify the presence of protected fragments at 492 bp and 342 bp, confirming the more abundant presence of the 164 and 120 isoform splice variants in penis mRNA. Again, this observation supports the RT-PCR experiments on the rat penis in which we found that the 164 and 120 splice isoforms appeared to be the most abundant VEGF splice variants present in rat penis mRNA (Fig. 2, A and B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our results show that several different mRNA splice variants of VEGF-A mRNA are expressed in both the rat and human penis and that there appears to be a distinct difference in the abundance of the various VEGF-A splice isoforms when compared to that in another rat tissue (rat lung) in which VEGF-A expression is known to be abundant. Whereas we have confirmed that the rat lung expresses most abundantly an mRNA encoding the 188 splice variant, the rat penis, in contrast, expresses the 164 splice variant of VEGF-A mRNA at greatest abundance. Other VEGF splice variants, including the previously described 120 splice variant, are also expressed at much lower levels by the lung and the penis. We were also surprised by our ability to detect the presence of the 144 VEGF-A splice variant, which was previously found only in placenta and some female reproductive tissues (uterus) [39–42]. Moreover, our sequencing studies of a large number of VEGF cDNAs obtained after RT-PCR amplification of rat penis mRNA revealed the presence of a new splice variant of VEGF-A that has the more unusual characteristic of retaining the first 3 exons (instead of the first 5) as well as having an apparent splice within the seventh exon. This splice variant is likely to be present in extremely low abundance, as we never detected a significant 330-bp amplification product in our RT-PCR experiments, nor did we ever find the corresponding protected exon fragments in our RNase protection experiments. While we considered the possibility that this unusual splice variant of VEGF might not be able to produce an angiogenic peptide, it is of interest that another rare splice variant of (mouse) VEGF mRNA has also been described that contains only the first 3 exons of VEGF. This splice variant (VEGF 115) has been shown to encode a peptide that is able to bind to VEGF receptor and activate mitogenesis of endothelial cells [46]. Therefore, the unusual VEGF splice variant (VEGF 110) we observed in our studies, although seemingly rare and low in abundance, probably has the potential to produce a functional VEGF peptide.
The recent approval by the Food and Drug Administration of Sildenafil citrate as the first oral therapy for erectile dysfunction has become a landmark for the development of pharmacotherapy for erectile dysfunction. However, all current pharmacologic treatments of erectile dysfunction consist of "on-demand" medications that produce temporary erections. At this time there is no curative treatment for erectile dysfunction. The work in this project is based on our conceptualization of the induction of vasculogenesis in the erectile tissue as a potential cure for the reduction of vascular elements that is frequently encountered in vasculogenic erectile dysfunction. In this descriptive study, VEGF-A mRNA expression was surveyed in the rodent and human penis. The present characterization of the various VEGF isoforms in erectile tissues is motivated by our interest in defining the angiogenic growth factors that regulate penile vasculogenesis and in potentially exploiting these findings for therapeutic applications to treat human impotence. One could conceivably consider the therapeutic application of VEGF peptides to stimulate further penile vascular development as has been contemplated for other vascular insufficiency conditions [9, 23]. Likewise, use of VEGF expression vectors provides a novel gene-therapeutic approach for the curative treatment of erectile dysfunction, an approach that is being contemplated for other gene products involved in the response [47, 48]. By identifying the predominant angiogenic peptides naturally expressed in the erectile tissues of the penis, we are one step closer to this goal.
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FOOTNOTES |
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2 Correspondence: Ridwan Shabsigh, Department of Urology, Columbia-Presbyterian Medical Center, Atchley Pavilion, 11th Floor, 161 Fort Washington Ave., New York, NY 10032. FAX: 212 305 0126;rs66{at}columbia.edu
Accepted: September 21, 1998.
Received: July 29, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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