Ex Vivo Expression of Angiogenic Growth Factors and Their Receptors in Human Penile Cavernosal Cells

Authors


Division of Urology, University of California San Diego Medical Center, 200 West Arbor Drive, San Diego, CA 92103-8897 (e-mail: mrajasek@ucsd.edu).

Abstract

ABSTRACT: Arteriogenic erectile dysfunction is associated with impairment of vascular perfusion to the erectile components of the penis. Animal studies have identified insulin-like growth factor (IGF-I) and vascular endothelial growth factor (VEGF) as penile angiogenic growth factors, but the role of these factors in humans is not well understood. We evaluated the ex vivo expression of IGF-I, VEGF, and their receptors (IGF-IR, Flt-1, and KDR) in human penile cavernosal smooth muscle cells (HCSMCs) to identify cellular and molecular pathways involved in the regulation of penile tissue vascularity. Primary culture was initiated with explants of human corpora cavernosa, and early passage (3–5) cells were used for these evaluations. Cultures were examined to verify the presence of smooth muscle cells and the absence of endothelial cell contamination. Specific monoclonal antibodies were used to localize growth factors and their receptors. To evaluate gene expression of VEGF, Flt-1, and KDR, total RNA was extracted from cavernosal cells and subjected to reverse transcriptase—polymerase chain reaction (RT-PCR) using custom synthesized primers. To study the effect on cell proliferation, 10 000 cells/well were exposed to varying concentrations of VEGF (0–50 ng/mL). At specified time periods the cells were trypsinized and counted. IGF-I and VEGF and their receptors were localized in the cultures, which were positive for the presence of smooth muscle cells and negative for endothelial cell contamination. RT-PCR evaluation revealed the expression of four splice variants of VEGF messenger RNA (VEGFs 121, 145, 165, and 189) and two of its receptors (Flt-1 and KDR). VEGF165 and VEGF121 were the most abundant forms of messenger RNA and Flt-1 appeared to be the most prominent receptor type in these cells. Exposure to VEGF elicited a twofold to threefold increase in the proliferation of HCSMCs. HCSMCs express both IGF-I and VEGF and their receptors, which may be important in the control of vascularity in human penile architecture.

Erectile dysfunction (ED) is defined as the consistent inability to attain and maintain a penile erection sufficient for satisfactory sexual performance. Although a wide range of risk factors contribute to the development of ED, vasculogenic ED is recognized as the most common etiology. Arteriogenic ED is associated with impairment of vascular perfusion to the erectile components of the penis (Christ, 1995). The cellular and molecular events involved in the regulation of penile tissue vascularity are not known. A clear understanding of these events would aid in the identification of newer therapeutic approaches to improve tissue vascularity and blood flow to the penis.

Several naturally occurring growth factors have been documented to induce angiogenesis and improve vascularity. These include the insulin-like growth factor-I (IGF-I; Delafontaine, 1995), the vascular endothelial growth factor (VEGF) family (de Jong et al, 1998), the fibroblast growth factor (FGF) family (Slavin, 1995), transforming growth factors (Jensen, 1998), and platelet-derived growth factor. Recent reports indicate a role for both IGF-I and VEGF in rat penile angiogenesis (Liu et al, 2001a,b). Of these, VEGF, a homodimeric glycoprotein of 34–45 kd, appears to play a pivotal role. Studies have shown that therapy with this growth factor is beneficial in vasculopathies associated with limb claudication (Baumgartner et al, 1998) and coronary artery disease (Lathi et al, 2001). At least four different VEGF transcripts resulting from alternative splicing of a single gene have been described in human cells. VEGF121 and VEGF165 are secreted as soluble compounds, whereas VEGF189 and VEGF206 remain associated with the cell surface or are primarily deposited in the extracellular matrix (Zachary, 2001). IGF-I exerts its biological function through high-affinity receptors (IGF-IRs), whereas VEGF acts through tyrosine kinase receptors on the cellular membrane, namely the kinase insert domain-containing receptor (KDR, also known as Flk-1), and the fms-like tyrosine kinase-1 receptor (Flt-1; Kroll and Waltenberger, 1998). The role of these growth factors and their receptors in human penile tissue vascularity is not known.

Human and other mammalian cultured arterial smooth muscle cells (SMCs) produce VEGF (Ferrara et al, 1991; Brown et al, 1997), which may constitute a local stimulus for angiogenesis or act as a permeability factor. Previous studies suggests that VEGF is abundantly expressed in rat and human penile tissues (Burchardt et al, 1999a,b). Corpora cavernosa are the main erectile bodies and cavernosal smooth muscle is the primary cellular component of the human penis (Andersson and Wagner, 1995). Although human cavernosal cells in culture exhibit characteristic features of SMCs (Krall et al, 1988; Dahiya et al, 1993), it is unknown whether these cells can synthesize IGF-I and VEGF during in vitro propagation. The present study was specifically designed to evaluate the expression of these growth factors under basal conditions and to localize their receptors in human cavernosal SMCs (HCSMCs) in primary culture. The effect of VEGF on HCSMC in vitro growth was also investigated.

Materials and Methods

Human Cavernosal Cell Culture

After institutional review board approval and informed patient consent were obtained, corporal tissue explants (1–2 mm3 in size) were collected from patients with diabetes undergoing penile prosthesis implantation. The fragments were placed into culture flasks containing growth medium (Dulbecco modified Eagle medium [DMEM] containing 20% fetal bovine serum [FBS]) and kept undisturbed for 4 days. The medium was exchanged with fresh growth medium, and this exchange was repeated every 4 days until the cultures became confluent. These primary cultures were examined for endothelial cell contamination using von Willebrand factor (vWF) as a specific marker and were characterized by specific immunochemical assays for α-actin and myosin expression to establish the presence of SMCs (Krall et al, 1988; Dahiya et al, 1993; Rajasekaran et al, 2001).

Indirect Immunofluorescence Assay for IGF-I, IGF-IR, VEGF, KDR, and Flt-1

Human cavernosal cells grown on glass chamber slides (70%-80% confluence) were washed in phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde for 30 minutes. The cells were incubated for 30 minutes with 5% FBS containing 1% Triton X-100 in order to block the nonspecific binding sites. The cells were further incubated overnight at 4°C with specific monoclonal antibodies for IGF-I, IGF-IR, VEGF (Santa Cruz Laboratories, Santa Cruz, Calif) (1:200) and specific monoclonal antibody for Flk-1 (Santa Cruz) (1:100) and polyclonal Flt-1 (Santa Cruz) (1:100) dissolved in PBS containing 1% FBS. In one set, cells were incubated with normal mouse immunoglobulin G (IgG; for IGF-I, IGF-IR, VEGF, and KDR)/rabbit IgG (for Flt-1) in the absence of primary antibody, which served as a negative control. After washing three times with PBS the cells were further incubated for 2 hours at room temperature with appropriate anti-mouse (for IGF-I, IGF-IR, VEGF, and Flk-1) or anti-rabbit (for Flt-1) secondary antibodies conjugated with either fluorescein isothiocyanate (FITC) or rhodamine. Incubation was terminated by washing with PBS, and the slides were mounted in Gel/Mount (Biomeda, Hayward, Calif). The slides that remained in darkness at 4°C were observed with a Leitz fluorescence microscope within 24 hours and photomicrographs were taken for evaluation (Rajasekaran et al, 1998).

RNA Isolation and Reverse Transcriptase—Polymerase Chain Reaction

Monolayer cells (80%-90% confluence) at passages 2–4 were harvested for RNA extraction and total RNA was isolated from these cells by the TriZol method (Gibco-BRL, Grand Island, NY). The quality and yield of the RNA were assessed by the 260:280-nm optical density ratio and by electrophoresis in 1.2% agarose gels containing formaldehyde and were viewed via ethidium bromide staining. Total RNA was subjected to reverse transcriptase—polymerase chain reaction (RT-PCR) using an Access RT-PCR system (Promega, Madison, Wis). Reverse transcriptase and PCR of a single target RNA was performed in a single tube using avian myeloblastosis virus RT (AMV RT) for first-strand DNA synthesis, and Thermus flavus (Tfl) DNA polymerase for second-strand cDNA synthesis and DNA amplification (5 μM oligo(dT), 10 mM dNTPs, and 1 mM Mg2+ in a volume of 50 μL). In selected tubes the RT was omitted to control for amplification from contaminating complementary DNA (cDNA) or genomic DNA. RT-PCR was performed in a DNA Thermal Cycler 480 (Perkin Elmer Cetus, Norwalk, Conn) for 45 minutes of RT at 48°C, 2 minutes of AMV RT inactivation, and RNA/cDNA/primer denaturation at 94°C. Amplification consisted of 40 cycles of 30 seconds denaturation at 94°C, a 60-second annealing step at 60°C, and a 120-second extension at 68°C.

The PCR products were size-fractionated by 1.2% agarose gel electrophoresis (agarose-1000, Gibco-BRL) and stained with 0.5 μg/mL ethidium bromide (Gibco-BRL) and the identity of the PCR products was confirmed. The primer sequences (Burchardt et al, 1999a,b; Ratcliffe et al, 1999) are shown in the Table. α-Actin was used as an internal control for RT-PCR reactions and the products were analyzed on a 1.2% agarose mini-gel system (Rajasekaran et al, 1998).

Cell Growth Assay

To study the effect of VEGF on the growth rate of HCSMCs, cells grown in a flask were trypsinized and counted using a hemocytometer, and resuspended at a concentration of about 106 cells/mL in DMEM-F12 supplemented with 0.1% bovine serum albumin (BSA). One-hundred microliters of the cell suspension were then aliquoted into eight wells of a multiwell plate, giving a total of about 10 000 cells per well. These cells were then exposed to varying concentrations of VEGF (0 to 50 ng/mL) in DMEM supplemented with 0.1% BSA. After 8 days of incubation the cells were trypsinized and counted. Experiments were repeated at least three times to confirm the findings.

Statistical Analysis

For cell growth assay, the data were expressed as means ± SEM and analyzed with the Student t test. A P value of < .05 was established as the criterion for statistical significance.

Results

Indirect Immunofluorescence Localization for Actin, Myosin, vWF, IGF-I, IGF-IR, VEGF, Flt-1, and Flk-1

Immunohistochemical studies confirmed the presence of smooth muscle cells in the cultures, which were devoid of endothelial cell contamination. Cells showed an intense staining for smooth muscle action and myosin, and they stained negative for vWF, the endothelial cell-specific marker (Figure 1, A-C). Positive fluorescence signals for VEGF (Figure 2A), Flt-1 (Figure 2B), IGF-I (Figure 2C), as well as IGF-IR (Figure 2D) were observed in these cells. The signal for VEGF was more intense and extensive than that of Flt-1. Differences in the distribution pattern were noted, with VEGF localized to the perinuclear intracytoplasmic region, whereas the Flt-1 receptor was localized to the cell membrane (Figure 2). A similar pattern of localization was observed with IGF-I (in the perinuclear intracytoplasmic region) and its receptor, IGF-IR (in the cell membrane). Cells were negative for KDR. In all controls only the fluorochrome background was observed, confirming the specificity of the staining.

Figure 1.

. Characterization of human cavernosal smooth muscle cells by immunofluorescence. (A) Actin, (B) myosin, (C) vWF, and (D) negative control (background). Magnification 40×.

Figure 2.

. Immunofluorescence label of IGF-I, IGF-IR, VEGF, and Flt-1 in human cavernosal cells. (A) VEGF, (B) Flt-1, (C) IGF, (D) IGF-IR. Magnification 40×. Arrows indicate cellular localization of IGF-I, IGF-IR, VEGF, and Flt-1 as described in “Results.”

Messenger RNA Expression for VEGF and its Receptors (KDR and Flt-1)

To evaluate the expression of VEGF and to explore the possible signal transduction pathways in HCSMCs, the messenger RNA (mRNA) expression of this growth factor as well as that of its receptors (KDR and Flt-1) was analyzed by RT-PCR. Specific oligonucleotide primers derived from the mRNA sequences of human endothelial VEGF or Flt-1/KDR were employed for PCR studies. The PCR products showed a length of 360–564 base pairs (bp) (VEGF; Figure 3, lanes 2–3) and 414 bp (Flt-1; Figure 3, lanes 4–5), respectively. The RT-PCR evaluation revealed the expression of four splice variants of VEGF mRNA (VEGFs 121,145, 165, and 189) and two of its receptors (Flt-1 and KDR) in HCSMCs. The major amplified species detected were fragments of 360 and 492 bp. These species correspond to the mRNA that encodes VEGF121 and VEGF165, respectively. Flt-1 appeared to be the most prominent receptor type in these cells, whereas KDR mRNA exhibited a relatively weak expression (421 bp; Figure 3, lanes 6–7).

Figure 3.

. Expression of VEGF splice variant mRNA in HCSMCs in primary culture. The mRNA was isolated, and following RT, PCR was carried out with specific primers for VEGF (lanes 2 and 3), Flt-1 (lanes 4 and 5), KDR (lanes 6 and 7). Data shown are from two representative experiments (n = 3). M, molecular weight marker (lanes 1 and 8).

Effect of VEGF on Cell Growth

Figure 4 shows the growth in HCSMCs after 8 days of exposure to different concentrations of VEGF (0–50 ng/mL). Cell proliferation at VEGF concentrations ranging from 2.5 to 37.5 ng/mL did not differ significantly from that in control wells exposed to nutrient mixture alone. Exposure to 50 ng/mL induced a significant (P < .05) increase in HCSMC growth at each time point (Figure 4).

Figure 4.

. VEGF165-stimulated HCSMC growth. HCSMCs (1 × 103 cells/well) were seeded into 8-well plates. VEGF was added at 50 ng/mL the following day. Cells were counted using a hemocytometer at the indicated times after VEGF addition. Values are expressed as mean ± SEM and P is in comparison with control cells exposed to growth media alone. Results of three independent experiments, each performed with quadruplicate wells per point.

Discussion

The penis is a vascular organ and the corpora cavernosa are dependent on adequate vascularity and blood flow (Andersson and Wagner, 1995). Recent reports indicate a role for angiogenic growth factors such as IGF-I and VEGF in the modulation of rat penile vascularity (Liu et al, 2001a,b). The most potent angiogenic growth factor (VEGF) and its tyrosine kinase receptor (Flt-1) were originally characterized in endothelial cells and have now been identified in a variety of cell types (Ferrara et al, 1991; Brown et al, 1997; Ishida et al, 2001). The results of our study demonstrate the production of both of these growth factors and the presence of their receptors in human cavernosal smooth muscle cells. In our study, RT-PCR evaluation showed four splice variants of VEGF mRNA, which is consistent with its expression in other SMCs. VEGF165, one of the abundant isoforms in HCSMCs, is considered the most active isoform (Muhlhauser et al, 1995). Flt-1, the predominant receptor in HCSMCs, has more than a 10-fold affinity for its ligand compared with that of the other VEGF receptor (KDR). Our demonstration of VEGF and Flt-1 in HCSMCs by PCR amplification and differential localization of VEGF to the perinuclear cytoplasm and Flt-1 to the cell membrane, respectively, by immunofluorescence suggests an autocrine role for this growth factor in penile cavernosal smooth muscle.

Differential expression of several growth factors involved in vasculogenesis, including VEGF, has been recently demonstrated in the penis of rats and humans (Burchardt et al, 1999a; Dahiya et al, 1999). Both IGF-I and VEGF have been shown to regulate proliferation and migration of rat cavernosal cells in culture (Liu et al, 2001a,b). Besides its angiogenic role, VEGF has been shown to regulate endothelial nitric oxide synthase (eNOS) expression in endothelial cells (Bouloumie et al, 1999). Intracavernosal injection of VEGF has been shown to induce the expression of eNOS as well as iNOS isoforms in rat cavernosal tissue (Lin et al, 2002). Additional biological actions of VEGF have been documented to include greater vascular permeability and vasodilation, which are inhibited by nitric oxide synthase inhibitors (Gavin et al, 2000).

To the best of our knowledge, this is the first demonstration of VEGF and its receptors in HCSMCs. The precise role of IGF-I, VEGF, and their receptors in human cavernosal smooth muscle is not clear. Our observation of VEGF-induced cell growth in HCSMCs supports the findings of Liu and associates (2001a,b) that this growth factor promotes proliferation of rat penile SMCs. Besides this role, it might also involve maintenance of smooth muscle integrity or regulation of SMC migration to proper sites in the cavernous space during vasculogenesis within the penis.

Conclusions

We have demonstrated that human cavernosal cell culture is a viable model for ex vivo localization and characterization studies, and that human cavernosal cells in culture express angiogenic growth factors as well as their receptors. The demonstration of these growth factors in HCSMCs and localization of their receptors by immunochemical studies suggest a possible in vivo autocrine role for these proteins. The question of whether these proteins are expressed in physiologically relevant quantities still remains to be addressed. Future investigations will examine this aspect by employing Western blot techniques to quantify the protein levels.

Footnotes

  1. A. Kasyan and W. Allilain were supported in part by AFUD summer scholarships.

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