1 Department of Urology, University of Washington School of Medicine and Harborview Medical Center, and 2 Center for Expression Arrays, University of Washington, Seattle, Washington
ABSTRACT
To investigate the full range of molecular changes associated with erectile dysfunction (ED) in Type 1 diabetes, we examined alterations in penile gene expression in streptozotocin-induced diabetic rats and littermate controls. With the use of Affymetrix GeneChip arrays and statistical filtering, 529 genes/transcripts were considered to be differentially expressed in the diabetic rat cavernosum compared with control. Gene Ontology (GO) classification indicated that there was a decrease in numerous extracellular matrix genes (e.g., collagen and elastin related) and an increase in oxidative stress-associated genes in the diabetic rat cavernosum. In addition, PubMatrix literature mining identified differentially expressed genes previously shown to mediate vascular dysfunction [e.g., ceruloplasmin (Cp), lipoprotein lipase, and Cd36] as well as genes involved in the modulation of the smooth muscle phenotype (e.g., Kruppel-like factor 5 and chemokine C-X3-C motif ligand 1). Real-time PCR was used to confirm changes in expression for 23 relevant genes. Further validation of Cp expression in the diabetic rat cavernosum demonstrated increased mRNA levels of the secreted and anchored splice variants of Cp. CP protein levels showed a 1.9-fold increase in tissues from diabetic rats versus controls. Immunohistochemistry demonstrated localization of CP protein in cavernosal sinusoids of control and diabetic animals, including endothelial and smooth muscle layers. Overall, this study broadens the scope of candidate genes and pathways that may be relevant to the pathophysiology of diabetes-induced ED as well as highlights the potential complexity of this disorder.
cavernosum; penis; Gene Ontology
ERECTILE DYSFUNCTION (ED) has origins associated with a complex interplay of factors ranging from psychosocial disorders (e.g., depression) to nerve injury (e.g., prostatectomy) to vascular pathologies (e.g., hypertension and atherosclerosis). Of all comorbid medical conditions, diabetes mellitus imparts the greatest risk of ED. Men with diabetes have a greater prevalence of ED and earlier onset of the condition compared with the general population (34, 101). Furthermore, men with diabetes present with more severe dysfunction and are less responsive to current pharmacological therapies for ED (14, 47, 91).
Penile erection is a complex and integrated neurovascular event (4). Because diabetes leads to neural and vascular pathologies, it has the potential to impact all components of the erectile response. Structurally, the erectile apparatus consists of paired organs (corpora cavernosa) that run the length of the penis. Each cavernosum is a specialized vascular compartment with sinusoidal trabeculae supporting a thick smooth muscle layer lined by endothelial cells. The arterial supply of the penis gives rise to helicine resistance arteries, which open directly into the sinusoidal spaces of the cavernosa (108). During erection, nitric oxide (NO) produced by nitrergic nerves [autonomic neurons containing neuronal NO synthase (nNOS)] and endothelial cells [endothelial NOS (eNOS)] stimulates relaxation of smooth muscle in the penile circulation and corpora cavernosa (108). In smooth muscle cells (SMCs), NO binds soluble guanylate cyclase, thus increasing cGMP levels and stimulating cGMP-dependent protein kinase I, which ultimately leads to relaxation (49). The ensuing arterial inflow and expansion of the cavernosa against the tunica albuginea (dense connective tissue surrounding the cavernosa) leads to compression of subtunical veins that impede blood outflow, producing a rigid erection.
Although recent studies have been instrumental in establishing mechanisms of impaired diabetic erectile function (for recent reviews, see Refs. 7 and 11), it is likely that the molecules and pathways identified to date represent only a small portion of the total changes occurring in erectile tissues that are due to diabetes. Thus the present study was designed to take a global approach to investigate the range of molecular changes in cavernosal tissue from animals with diabetes-associated ED. The ultimate goal was to advance our understanding of the pathogenesis of diabetes-associated ED and to help provide a rationale for novel treatments and preventive therapies.
METHODS
Animals and induction of diabetes.
Male Fischer-344 rats (Taconic Farms) were housed in groups of two or three in a specific pathogen-free environment. Rats were maintained in a temperature-controlled room with a 12:12-h light-dark cycle and received food and water ad libitum. All procedures were approved by the Animal Care and Use Committee of the University of Washington and were performed in accordance with National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. At 2 mo of age, rats were separated into individual cages and injected intraperitoneally with 35 mg streptozotocin (STZ; Sigma Chemical)/kg body wt in sterile citrate buffer to induce diabetes. Control rats were injected with an equivalent volume of citrate buffer. Twenty-four hours after the injections, blood glucose was checked via a tail stick using an Accu-Check glucose monitor (Roche Diagnostics). Animals were considered diabetic if their blood glucose levels were above 300 mg/dl. Body weight and glucose levels were monitored weekly in all animals over the course of the study (10 wk). The 10-wk duration of diabetes was selected based on previous studies demonstrating significantly reduced erectile responses in Fischer-344 rats after 812 wk of diabetes (26, 98). Values are presented as means ± SE. Comparison between two means was performed using Student's unpaired t-test.
Measurement of erectile responses.
Ten weeks after the induction of diabetes, intracavernosal pressure (ICP) changes in response to cavernous nerve stimulation were measured in control (n = 8) and diabetic rats (n = 6) as described previously (125). Briefly, anesthesia was induced with 5% isoflurane (Novaplus) vaporized in 100% O2 and then maintained with 1.5% isoflurane during the procedures. The right carotid artery was cannulated to monitor arterial blood pressure. The penis was exposed, and the corpus spongiosum was mobilized to facilitate insertion of a 25-gauge needle into the corpus cavernosum. The needle was attached via polyethylene-50 tubing to a pressure transducer (Kent Scientific) filled with heparinized saline. To establish baseline parameters, the cavernous nerve was stimulated (6 V, 10 Hz, 1 min) with a bipolar electrode, Grass S48K nerve stimulator, and stimulus isolation unit SIU5 (Grass Telefactor). Pressure changes were recorded continuously in response to 1-, 2-, 4-, 6-, and 10-V stimulations, each for a duration of 1 min with 5 min between voltages. ICP and arterial pressure were converted to analog signals and transmitted to a data-acquisition program (Hem 3.2, Notocord). The erectile response was calculated using the area under the curve (in mmHg) for ICP during the 1-min stimulation period (ICP). This value was divided by the area under the curve for the calculated mean arterial pressure (MAP) during the same 1-min stimulation (
ICP/MAP). Groups were compared by one-way ANOVA with a Student-Newman-Keuls multiple-comparison test.
Tissue collection.
Immediately after the ICP measurements, the penis was rapidly dissected free at the level of the crura. After removal of any overlying skeletal muscle, equal portions of the crura and shaft were placed into1.5-ml tubes, flash frozen in liquid nitrogen, and then stored at 80°C. For histology, a small cross section of the proximal shaft was suspended in OCT compound (Tissue-Tek), frozen over liquid nitrogen, and stored at 80°C. An independent cohort of control and diabetic Fischer-344 rats (n = 5 each) was generated and used to collect penile protein samples for Western blot analysis. The samples were collected as described above and stored at 80°C.
RNA isolation.
Total RNA isolation was performed using a combination of TRIzol reagent (Invitrogen) and RNeasy columns (Qiagen). Penile tissue stored at 80°C was gradually thawed using RNAlater-ICE (Ambion) as described by the manufacturer. Next, tissue was homogenized in TRIzol reagent according to the manufacturer's instructions with the following modifications. After the addition of chloroform, centrifugation was performed using Phase Lock Gel Heavy 2-ml tubes (Eppendorf) to improve recovery of the aqueous phase of the solution. One half volume of 100% ethanol was combined with the recovered aqueous solution, and the mixture was added to an RNeasy minicolumn. This and all subsequent steps were performed as described by the manufacturer [Qiagen RNeasy handbook (3rd ed.)]. On-column DNase digestion was done using the Qiagen RNase-Free DNase kit as directed in the RNeasy handbook (Appendix D). RNA quality was examined by the RNA 6000 LabChip Kit on the 2100 bioanalyzer (Agilent Technologies). Quantity and absorbance at 260/280 nm of total and cRNA was assessed by an ultraviolet spectrophotometer. After quantification, RNA samples were divided into 5-µg aliquots and stored at 80°C.
Microarray hybridization and data analysis.
Double-stranded cDNA was synthesized from total RNA, amplified as cRNA, labeled with biotin, and hybridized to Affymetrix Rat 230A GeneChips, which were washed and scanned at the University of Washington's Center for Expression Arrays according to procedures developed by the manufacturer. A total of 5 control and 5 diabetic samples were used requiring a total of 10 GeneChips. Image processing was done using Affymetrix GCOS software. The quality of hybridization and overall chip performance was determined by visual inspection of the raw scanned data and the GCOS-generated report file. Raw data were loaded into the Rosetta Resolver Gene Expression Data Analysis System (Rosetta Biosoftware; Seattle, WA) for analysis using the Rosetta Resolver System error model (97). On the basis of five biological replicates from each experimental condition, Resolver was used to generate 1) P values to determine whether genes were detected (i.e., present call); 2) P values from ANOVA to establish differentially expressed genes between the control and diabetic groups; and 3) an estimate of fold change for each gene relative to control. These parameters were used for statistical filtering of the array data as described in the RESULTS. The Resolver output values for those genes selected as significant are available in the online supplement (Supplemental Figs. IIII; available at the Physiological Genomics web site).1
In compliance with MIAME standards, Affymetrix data files (CHP and CEL) have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) repository (query Accession No. GSE2457). The gene symbols used in this study are, to the best of our knowledge, those designated on NCBI Entrez Gene, and every effort was made to avoid the use of ambiguous gene designations. Also, Affymetrix Rat 230A probe set identifiers were annotated via the NetAffx Analysis Center (www.affymetrix.com) in early 2005. Because the annotations are periodically updated, changes in Affymetrix Probe Set gene designations are possible. GeneChip hybridizations, scanning, and Resolver analysis were performed at the Center for Expression Arrays at the University of Washington.
Real-time PCR.
With the use of the samples from the array experiments (n = 5 for each group), 2 µg of total RNA were reverse transcribed into cDNA using the RETROscript first-strand synthesis kit (Ambion). Each cDNA sample was then used as a template for real-time PCR amplification using SYBR green PCR Master Mix (Applied Biosystems) and forward/reverse primers for the various target genes (see on-line supplement for primer sequences). Amplification and detection was performed on an Applied Biosystems 7900 Real-Time PCR system according to the manufacturer's instructions using a two-stage cycle of 95°C for 15 s and 62°C for 1 min repeated for 40 cycles followed by a dissociation stage. Threshold cycle (CT) values were exported into spreadsheets, and relative changes in gene expression were then calculated using the method as described previously (73). Results are given as the fold change relative to control (nondiabetic) cDNA while using ß-actin (Actb) gene expression as a reference gene. All samples were prepared and examined in parallel. Conventional, nonquantitative PCR was performed for ceruloplasmin (Cp) to confirm the presence of its two splice variants based on previously published primer sets (20, 89). A common forward primer was used for both splice variants of Cp, 5'-gta tgt gat ggc tat ggg caa tga-3'. The Cp serum or secreted form was detected using the reverse primer 5'-tca tct gtc cat cgg cat ta-3', which yields a product size of 374 bp. The glycosylphosphatidylinositol-anchored form (Cp-GPI) was detected using the reverse primer 5'-cct gga tgg aac tgg tga tgg a-3', which yields a product size of 449 bp. cDNA template was amplified using a Qiagen Taq PCR core kit on an Eppendorf Mastercycler with the following cycle parameters: 95°C for 15 s, 57°C for 45 s, and 72°C for 1 min (repeated for 35 cycles).
Western blot analysis.
Cavernosal tissue was weighed, minced with a razor blade, and homogenized for 30 s in 10 vol of ice-cold lysis buffer containing fresh protease inhibitors. After centrifugation at 16,000 g for 20 min at 4°C, the supernatant was collected, and protein concentrations were determined using the MicroBCA kit (Pierce). Equal amounts of protein (50 µg) were run on precast polyacrylamide gels (GeneMate) and transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was blocked for 1 h at room temperature in 5% nonfat milk in 1x PBS containing 0.1% Tween 20. Membranes were incubated for 1 h at room temperature with primary goat polyclonal anti-human CP antibody (Sigma, C0911, 2 µg/ml) diluted in blocking solution. The primary antibody was detected with horseradish peroxidase-conjugated anti-goat secondary antibody (Amersham Biosciences), diluted 1:1,000 in blocking solution, and incubated for 1 h at room temperature. Blots were developed using enhance chemiluminescence reagents (Amersham Biosciences), and the results were quantified by densitometry (LabWorks). Membranes were incubated with mouse monoclonal anti-ß-actin antibody (Sigma, AC-15, 1:40,000 dilution) as a reference for protein loading. Values represent CP densitometry normalized to ß-actin and are presented as means ± SE. Comparison between means was performed using Student's unpaired t-test.
Histology and immunohistochemistry.
Frozen penile sections (8 µm) were cut from the OCT-embedded tissue blocks and placed on glass slides. For immunohistochemistry, slides were fixed in cold acetone (20°C) for 5 min, air dried, and then placed in PBS. As previously described, standard immunohistochemistry techniques were used to identify antigens of interest (126). Incubation with primary antibody was done for 1 h at 37°C in a humidified chamber. Secondary antibody was applied for 30 min at room temperature. For colocalization staining (e.g., CP and PECAM/CD31), primary and secondary antibodies were applied sequentially as described above and then repeated for the next antibody pair. The primary antibodies used and working concentrations were as follows: mouse monoclonal anti-rat CD31 (Chemicon, 1393Z, 20 µg/ml) and goat polyclonal anti-human CP (Bethyl Laboratories, A80-124A, 5 µg/ml). The secondary antibodies used and concentrations were as follows: rabbit anti-mouse IgG Alexa 488 and donkey anti-goat Alexa 568 (Molecular Probes, 10 µg/ml). Slides were mounted and coverslipped using Gel Mount (Biomeda). Fluorescent images were captured using a Nikon Eclipse E600 microscope integrated with a RT color Spot charge-coupled device camera (Diagnostic Instruments). Image processing, such as the merging of dual-labeled sections, was done with Spot Advanced software (Diagnostic Instruments).
RESULTS
Glucose levels in diabetic rats.
The mean body weight of diabetic rats at week 10 was significantly lower than that of controls (245 ± 8 vs. 377 ± 13 g, respectively, P < 0.01). Blood glucose was significantly higher in diabetic rats relative to control (386 ± 8 vs. 94 ± 5 mg/dl, respectively, P < 0.0001), and glucose levels in the STZ-treated group were maintained at 300 mg/dl or above throughout the course of the study.
ICP responses to nerve stimulation demonstrate ED in diabetic rats.
At 10 wk after the induction of diabetes, there was decreased erectile function in diabetic animals in response to cavernous nerve stimulation (Fig. 1). The mean ICP/MAP (area under the curve for the ICP and MAP tracings during the 60-s stimulation, in mmHg·s) was lower at all voltages in the diabetic group compared with control (statistically significant at 4, 6, and 10 V, P < 0.01). This confirmed the presence of diabetes-associated ED in the STZ-treated animals, and the observed reductions in ICP/MAP (
30%) are consistent with previously reported values of ICP responses in Fischer-344 diabetic rats, which showed
2540% reductions in ICP depending on nerve stimulus parameters (98). El Sakka et al. (26) reported much larger reductions in maximum ICP, which were
75% lower in diabetic Fischer-344 rats. Examples of the raw ICP and arterial pressure tracings for a representative diabetic and control animal are shown in Supplemental Fig. I. Maximum ICP/MAP was also significantly lower in diabetic rats versus controls (data not shown).
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Various strategies were used to examine the 529 changed genes (350 were annotated genes or homologs) to determine their possible biological relevance to diabetes-associated ED. In particular, the Gene Ontology (GO) tool GoMiner was utilized to categorize genes according to biological process, molecular function, and subcellular localization (130). On the basis of GoMiner analysis, there was a significant enrichment of downregulated genes in GO categories such as the following (no. of genes downregulated in diabetes per GO catergory/no. of genes in category on GeneChip): extracellular matrix (ECM) structural constituent (11/39), fibrillar collagen (7/9), ECM structural constituent conferring tensile strength (6/10), skeletal development (13/98), and ossification (5/37). Also, there was an enrichment of upregulated genes in categories such as fatty acid metabolism (12/93), fatty acid oxidation (5/17), and response to oxidative stress (5/44). The entire GoMiner output file (including GO identifiers, P values, and GO terms) is available in the on-line supplement. Table 1 shows groupings of genes based on similar GO and functional categorization. Many genes are represented by more than one Affymetrix probe set on the RAE230A GeneChip, and, in many cases, multiple probe sets for the same gene were found to be significant [e.g., chondroitin sulfate proteoglycan 2 (Cspg2), lysyl oxidase (Lox), and others, as shown in Table 1]. The functional grouping of the differentially expressed transcripts shows several elastic fiber-related genes that were downregulated in the diabetic cavernosum [e.g., Cspg2, Lox, fibrillin 1 (Fbn1), and elastin (Eln)]. Similarly, genes encoding a total of eight different collagen family members were downregulated in the diabetic samples. Various other ECM-related genes were differentially expressed in the diabetic cavernosum, including genes [secreted phosphoprotein 1 (Spp1), tenascin C (Tnc), and secreted acidic cysteine-rich glycoprotein (Sparc)] that encode members of the functionally grouped matricellular proteins (Table 1).
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Real-time PCR confirmation of array results.
We used real-time PCR to examine expression for 23 genes shown to be significantly different by array analysis. These genes were selected based on fold change in expression, GO results, and/or potential roles in diabetic complications or vascular biology as identified via PubMatrix. Table 2 shows the fold change estimated by Resolver for the GeneChip data compared with the fold change determined using real-time PCR. The majority of genes showed similar changes in expression when the two methods were compared. One exception is elastase 2 (Ela2), which showed no difference between groups by real-time PCR but had an estimated 4.5-fold change by Resolver analysis. Closer examination of the Resolver data showed very low-intensity values for this Affymetrix probe set (1387471_at) in the control and diabetic groups. The difficulties of detecting changes in transcripts with very low signal intensity by microarray has been established, and this difficulty likely caused Ela2 to be falsely called significant (97). Vacuolar H+-ATPase subunit M9.2 was selected as an unchanged gene between the diabetic and control groups, and this lack of difference was confirmed by the real-time comparison. Overall, the genes examined by real-time PCR showed good agreement with the Resolver estimates of fold change.
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The present analysis provides a global view of the effect of diabetes on erectile tissue at the mRNA level. This is the first study to use microarrays to examine gene expression in cavernosal tissue from animals with diabetes-associated ED. The identification of 500 differentially expressed genes/transcripts in the diabetic samples underscores the complex nature of this condition and supports the notion that diabetes causes multiple pathophysiological changes in erectile structures of the penis. Determining biological relevance of so many altered genes poses a tremendous challenge. We used several strategies for biological interpretation of our results such as GO analysis and mining of NCBI literature. It is not surprising in diabetes that a large portion of the significantly upregulated genes are involved in pathways required for lipid metabolism. A number of the differentially expressed genes have been previously associated with diabetic complications in other organ systems (see Table 2 for a few examples). This suggests the existence of common molecular pathways underlying the development of diabetic complications in different tissues. Many of the significantly changed genes in our study have no reported role in diabetes and no clear function related to erectile physiology.
Collagen, elastin, and ECM genes.
The GO analysis highlights the considerable downregulation of ECM genes in the cavernosal tissue of diabetic rats. The ECM of human and rat erectile tissues is composed mainly of collagen, elastic fibers, and various proteoglycans (43, 52, 74, 96). Several studies examining human erectile tissues have demonstrated qualitative and quantitative changes in collagen and elastic fibers in the cavernosum of impotent patients (21, 95, 105, 128). Similarly, animal studies show morphological and ultrastructural changes in the cavernosum of diabetic and aged rats (15, 30, 102). To date, the specific pathways and molecules mediating such changes have not been fully elucidated. Mathematical modeling of penile hemodynamics suggests that cavernosal expandability and tunical distensability are important factors contributing to erection (40, 118). Accordingly, diabetes-induced changes in matrix components and ECM structural organization of the tunica albuginea and/or corpus cavernosum could act to impair erectile function. The present array analysis provides several prospective molecules that could be contributing to alterations in cavernosal structure associated with diabetes.
The concurrent downregulation of genes encoding components of collagen type 1, 3, 5, 6, and 11 (listed in Table 1) certainly suggests that alterations in collagen biosynthesis and/or composition may contribute to the development of ED in this model. We observed decreased diabetic expression of Col1a1 and Col1a2, both of which encode fibrils forming collagen type 1, which provides tissue with tensile strength and stiffness (41). Collagen type 1 is the most abundant form of collagen in human and rat erectile tissues and forms the bulk of the matrix structure of the cavernosum (74, 96). The Col3a1 gene, which was reduced twofold in the diabetic cavernosum, encodes a collagen component of reticular fibers in elastic tissues such as skin, the lung, and blood vessels (41). Additionally, all three collagen type 5 -chain genes (Col5a1, Col5a2, and Col5a3) were reduced in the diabetic samples. Collagen type 5 typically forms heterotypic or mixed fibrils with collagens types 1 and 3 (41).
Elastic fibers, composed of microfibrillar bundles with an inner core of cross-linked elastin, provide tissues such as the corpora cavernosa with resilience and deformability that allow repeated cycles of expansion and recoil. The array analysis revealed significantly reduced expression of several genes encoding microfibril and elastic fiber-associated molecules in the diabetic cavernosum (Table 1). Eln and Fbn1 were modestly downregulated. Fibrillin protein is a principal component of elastin-associated microfibrillar bundles (for a recent review, see Ref. 62). Diabetes was associated with decreased microfibrillar-associated protein 2 (Mfap2) and biglycan (Bgn) gene expression. Mfap2 encodes a protein that may be important in microfibril structural integrity, whereas biglycan protein can form a complex with both MFAP2 and elastin molecules (62). Cspg2 (alias versican), which had a two- to threefold reduced gene expression in diabetic samples, binds fibrillin molecules and may act to link elastin-associated microfibrils with surrounding ECM components in tissue (55). Interestingly, Cspg2 overexpression in cultured aortic SMCs increased elastin mRNA levels and induced elastic fiber formation (79). Merrilees and colleagues (79) also showed that Cspg2 overexpression promotes elastic fiber deposition in balloon-injured rat carotid arteries.
The observed decline in diabetic gene expression of Lox is noteworthy considering that Lox encodes an enzyme required for proper cross-linking of elastin and collagen in vascular tissue (50, 75). LOX cross-linking activity mediates the stabilization of collagen and elastin fibers in the ECM and also helps deposit elastin onto microfibrils (58). In fact, arteries of Lox knockout mice show considerable structural alterations including fragmented elastic fibers (50, 75). Interestingly, low-density lipoprotein (LDL) decreases Lox mRNA in cultured aortic endothelial cells, and hypercholesterolemia is associated with downregulation of Lox expression in aortic tissue (100). Lox mRNA is detected in both endothelial cells and SMCs in vitro, and so multiple sources of LOX are likely in the cavernosum (100, 109). The levels of LOX or perhaps even the related LOX-like proteins (i.e., LOXL1LOXL4) may be important in cavernosal biology that is particularly related to ECM assembly, maintenance, and remodeling.
Previous studies evaluating human cavernosal structures show reduced elastic fibers in samples from impotent versus potent patients and diabetic versus nondiabetic patients (105, 128). Similar evaluations of elastic fibers in erectile tissue using animal models of diabetes have not been reported. Salama and colleagues (102) described thickening of collagen bundles in the diabetic rat tunica albuginea and altered collagen architecture, but changes in elastic fibers were not addressed. Interestingly, the tunica albuginea of aged rats shows structural alterations including thinning and fragmentation of elastic fibers (15). These observations are consistent with studies of the vasculature showing that diabetes is associated with reduced elasticity of coronary arteries and decreased elastin levels in the aorta (68, 112).
Another notable finding is the reduced expression of Sparc (alias osteonectin), Spp1 (alias osteopontin), and Tnc in the cavernosum of diabetic rats. Each of these genes encodes a member of the matricellular family of proteins. Matricellular proteins are unique ECM molecules that do not appear to have direct structural roles but instead mediate cell-matrix interaction and cell function. For instance, Spp1 and Sparc are reported to influence diverse biological processes including vascular function and structure (refer to Table 2). Carotid arteries of Spp1 knockout mice exhibit loosely organized collagen fibers, and vessels have increased compliance (81). Similarly, matrix disorganization and smaller diameter collagen fibrils are observed in healing wounds of Spp1 knockouts (71). Sparc knockout mice also have phenotypes involving alterations in ECM organization and structure. For example, collagen fibers surrounding tumors grown in Sparc knockouts have reduced fiber diameter and attenuated fiber cross-linking (13). The dermis of mice lacking Sparc has decreased collagen fibril size and altered collagen composition, suggesting the presence of immature collagen fibers (12). It remains to be determined whether reduced matricellular protein expression in the diabetic cavernosum is causing alterations in ECM production and/or assembly.
Ceruloplasmin upregulation in the diabetic cavernosum.
As explained in the RESULTS, we used PubMatrix searches of differentially expressed genes to help identify molecules that might influence erectile responses by altering vascular function. CP is an example of one such molecule previously shown to affect vascular responses. CP is a multifunctional protein, initially isolated from plasma, which has numerous proposed biological functions including copper transport, iron metabolism, and substrate oxidation and reduction (10). Alternative splicing of the Cp gene yields two distinct mRNA sequences that encode GPI-linked (membrane anchored) and secreted forms of CP, both of which were upregulated in the diabetic cavernosum based on real-time PCR (89). This is consistent with a recent study (42) showing increased Cp gene expression (3.1-fold, Affymetrix data) and elevated CP protein (1.7-fold) in retinal cells from STZ-treated rats. Previous studies (2, 23) have reported increased serum CP levels in patients with Type 1 or 2 diabetes.
CP is expressed in numerous tissues including the liver, testis, lung, brain, placenta, and eye (3, 65). Vascular SMCs grown in vitro express Cp mRNA (19), as do cultured SMCs derived from human cavernosal tissue (unpublished observations, data not shown). Strong CP antibody labeling of cavernosal SMCs is consistent with the idea that SMCs are the primary source of Cp gene and CP protein expression in our samples. Previous examinations of liver and lung tissue showed lack of Cp mRNA in the vascular endothelium, making it unlikely that cavernosal endothelial cells are a source of CP (65). However, liver endothelial cells are able to bind and internalize CP from the serum, possibly through an interaction with a membrane receptor (59, 115). Therefore, CP localized to the cavernosal endothelium (Fig. 3, C and E) could be from the binding of CP produced locally (e.g., SMCs) and/or secreted by the liver (i.e., circulating in blood). The 16-fold upregulation of Cp serum mRNA in diabetic samples seems to suggest that CP secretion may be enhanced locally within the erectile tissues. The Cp-GPI splice variant was also upregulated by diabetes (2.4-fold); based on mRNA abundance, this variant appears to be the principal form of Cp mRNA normally expressed in the cavernosum. Interestingly, GPI-anchored proteins exhibit cell to cell transfer in certain experimental conditions (67). Regardless of the source or form, higher amounts of CP protein were detected in penile tissues of diabetic rats, and the most prominent CP staining was localized to the cavernosal SMCs and endothelium.
In terms of vascular function, CP at physiological levels impairs endothelial-dependent relaxation of the aorta in a dose-dependent manner (17). This reduced vascular reactivity appears to be due to the ability of CP to decrease endothelium-dependent NO production via inhibition of eNOS (9). These activities may be related to enhanced Cu2+ transfer to vascular cells given that CP mediates the transfer of Cu2+ across cell membranes and that Cu2+ loading is observed in CP-treated endothelial cells (9, 92). Interestingly, Cu2+ and other divalent transition metals have direct inhibitory effects on nNOS activity through direct binding of metal ions to the NOS enzyme (93, 94). Thus CP and/or CP-derived Cu2+ can inhibit the two primary sources of NO required for penile erection (nNOS and eNOS). Additionally, CP has the ability to catalyze oxidation of NO to highly reactive nitrosonium (NO+), which may directly alter NO bioavailability in the penis (10). Future studies are required to determine whether the association between Cp expression and erectile function is causal.
Lipoprotein lipase expression.
According to the Resolver analysis and real-time PCR, Lpl gene expression was upregulated nearly threefold in diabetic penile tissue. Previous reports of changes in Lpl expression in diabetic animals are variable depending on the type of tissue examined (25, 103, 114). The Lpl gene codes for LPL protein, which is expressed in multiple tissues including the heart, muscle, adipose tissue, and vascular smooth muscle. LPL has the enzymatic function of triglyceride hydrolase and the nonenzymatic role of a ligand/bridging factor for lipoprotein uptake. In the vasculature, SMCs are positive for Lpl mRNA and protein, whereas endothelial cells have only LPL protein (16, 57). Immunofluorescence of control and diabetic cavernosal tissue showed strong LPL localization in the cavernosal sinusoids and adjacent vessels (Supplemental Fig. II).
Transgenic mice overexpressing human Lpl in vascular smooth muscle have augmented contractile responses and reduced endothelial-dependent relaxation in the aorta (27). The aortas from these transgenic Lpl mice have increased levels of free fatty acids (FFAs). This suggests that LPL within vessels acts to liberate FFAs that can then be taken up by vascular cells. FFA overloading in SMCs and endothelial cells can enhance the generation of reactive oxygen species, which reduces the production and bioavailability of NO (28, 53). Also, LPL activity may alter vascular function by increasing cellular uptake of modified LDLs [e.g., oxidized LDL (oxLDL) and glycated LDL] by SMCs and endothelial cells (6, 80, 86, 131). oxLDL can impair both vascular and cavernosal relaxation, whereas glycated LDL has been shown to induce endothelial cell apoptosis and reduce eNOS expression (1, 5, 107). These studies present multiple mechanisms by which cavernosal upregulation of Lpl could contribute to ED.
Cd36 expression.
The twofold upregulation of Cd36 gene expression in the diabetic cavernosum is consistent with previous studies (45, 90) demonstrating increased CD36 in the heart of diabetic mice and tissues from diabetic rats. Interestingly, macrophage Cd36 gene expression is increased by glucose, and atherosclerotic lesions from patients with hyperglycemia have greater CD36 levels (46). CD36 (also termed fatty acid translocase) is a type B scavenger receptor that binds to collagen, thrombospondin, anionic phospholipids, and oxLDL (for a review, see Ref. 82). CD36 is expressed by a variety of cell types including cardiac myocytes, skeletal myocytes, microvessel endothelial cells, and cultured aortic SMCs (45, 60, 99, 113). Immunostaining for CD36 in the diabetic and control rat cavernosum showed the strongest staining in capillaries and microvessels surrounding and within erectile tissues as well as discontinuous staining of the cavernosal sinusoids (Supplemental Fig. III).
Studies of mutant mice have established a physiological role of CD36 in fatty acid and lipoprotein metabolism (32). CD36-deficient cells show decreased binding and uptake of oxLDL. In cultured aortic SMCs, CD36 mediates the cellular internalization of oxLDL (99). In apolipoprotin E (apoE)/Cd36 double knockouts, the loss of CD36 is associated with reduced atherosclerotic lesion formation and preserved endothelium-dependent vessel responses (33, 64). The presence of CD36 in hypercholesterolemic apoE-null mice alters eNOS localization, which then impairs NO production (64). Increased CD36 levels in the penis could adversely affect cavernosal relaxation through mechanisms involving oxLDL uptake and/or direct interaction with eNOS.
SMC biology and phenotype.
Vascular SMCs show evidence of a spectrum of differentiated phenotypes, and adult SMCs are capable of dynamic shifts in phenotype depending on local signals such as growth factors, matrix interactions, inflammatory stimuli, and hemodynamic forces (for a review, see Ref. 88). Importantly, diabetes is a condition that induces phenotypic modulation of SMCs, as evidenced by the expression of embryonic forms of matrix and contractile genes in aortas of diabetic rats (37). In culture, SMCs isolated from diabetic animals have increased growth rates, altered contractile protein expression, and different ultrastructural organization compared with nondiabetic SMCs (29). Similarly, SMCs derived from vessels of diabetic patients display phenotypic differences, including increased proliferation and migration as well as distinct cell morphology (31). Given that 1) diabetes alters the SMC phenotype, 2) erectile tissues are specialized SMC-enriched vascular structures, and 3) normal SMC function is prerequisite for proper erectile activity, we sought to identify those differentially expressed genes in diabetic samples with the potential to impact SMC biology and phenotype.
The increased diabetic expression of transcription factors from the Kruppel-like factor family (Klf5 and Klf15, increased 3- and 2-fold, respectively) could be indicative of alterations in the SMC phenotype. Klf5 (alias BTEB2) is expressed in the medial SMC layer of the neonatal aorta but not in the adult aorta (122). Also, Klf5 upregulation is localized to intimal SMCs of restenotic lesions and injured vessels, suggesting that Klf5 plays a role in phenotypic modulation of SMCs in vascular disease (51, 122). Specifically, it appears that Klf5 acts as a transcriptional regulator of nonmuscle myosin heavy chain IIB (Myh10; alias SMemb) expression in SMCs, which is indicative of a dedifferentiated, proliferative SMC phenotype (122). Although Myh10 was unchanged in our study, we observed upregulation of the related gene myosin heavy chain polypeptide 9 (Myh9; alias nonmuscle myosin heavy chain IIA) in diabetic cavernosal tissues (2-fold increase, P = 0.0008). Myh9 expression has been detected in primary and restenotic atherosclerotic lesions (84). Additionally, MYH9 protein levels increased progressively in the vessel media and neointima after carotid injury in rats (38). The role of Klf15 in SMC biology has not been studied, and so the significance of its upregulation in the diabetic cavernosum is unknown. Klf15 expression is present in all muscle lineages, including SMCs of the vasculature (44).
In relation to the SMC phenotype, it is important to point out that we did not detect diabetes-associated changes in several SMC differentiation marker genes (e.g., smooth muscle -actin, smooth muscle myosin heavy chain, calponin 1, and smoothelin). This would seem to contradict the presence of profound or widespread dedifferentiation of SMCs in the diabetic cavernosum. However, a previous array analysis of vascular neointima formation, characterized by SMC phenotypic modulation and proliferation, did not demonstrate wholesale changes in the above-mentioned SMC markers (39). Also, we cannot rule out changes in the expression of various SMC genes at times other than our 10-wk end point or that alterations were present at the protein but not mRNA level.
On the basis of literature mining, we were able to identify additional genes that could be affect SMC biology in the diabetic rat penis. For example, diabetic upregulation of C3 (2-fold increase, P = 0.00345) may alter cavernosal SMC tone given that C3 is able to cause constriction of isolated arteries (77). C3 expression was detected selectively in the aorta and cultured SMCs from spontaneously hypertensive rats (72). Furthermore, exogenous C3 protein caused dedifferentiation of SMCs and shifted gene expression toward a synthetic SMC phenotype (72). Another upregulated gene in the diabetic cavernosum, Cx3cl1 (alias fractalkine) encodes a protein that is able to increase SMC adhesion and proliferation in an autocrine fashion in response to inflammatory cytokines (18). Cx3cl1 mRNA and protein are upregulated in diabetic rat kidneys as well as in atherosclerotic arteries from patients with and without diabetes (63, 127). Finally, we detected significantly reduced gene expression for S100a4 (alias metastasin and calvasculin), which encodes a member of the S100 family of Ca2+-binding proteins. S100a4 has been implicated in various cellular processes and appears to be constitutively expressed in the aorta and cultured SMCs (24). Cellular localization of S100A4 protein in SMCs and vascular tissue shows S100A4 to be strongly associated with actin stress fibers and with the sarcoplasmic reticulum (76). In addition to interacting with intracellular proteins, S100A4 may have an extracellular function as a secreted protein (124).
The potential for phenotypic modulation of the diabetic cavernosal smooth muscle is an intriguing idea that has yet to be established. Changes in any number of the above genes may be indicative of an altered differentiation/maturation state in SMCs of the cavernosum and penile vasculature that would have important implications on erectile function. Phenotypic modulation may well reflect changes in the assortment of necessary contractile proteins, signaling molecules, growth factors, and matrix modulators of properly functioning cavernosal SMCs.
Limitations.
Expression analysis with microarrays can be a valuable and powerful technique, but certain limitations exist in the present study that merit discussion. Although erectile tissues are relatively SMC enriched, there is clearly a mixture of cell types in the harvested cavernosal samples. As a result, all the various cell types potentially contribute to the differential gene expression detected in this analysis. With the exception of certain cell type-specific genes, further characterization will be required to identify a particular source of altered gene expression. Additionally, our analysis merely establishes an association between the observed gene changes and diabetic ED. Further experimentation is necessary to distinguish between differentially expressed genes that are actual mediators of ED, markers of ED, or inconsequential to ED. This is an expected challenge often present in microarray studies, and so our analysis should be primarily viewed as hypothesis generating. Also, the Affymetrix RAE 230A GeneChips contain probe sets representing over 15,000 genes and transcripts including the majority of full-length, well-annotated rat genes. However, the 230A GeneChip does not contain probe sets for all rat genes, and, as a result, this analysis is incomplete. Furthermore, evaluation of gene expression at a single time point may miss critical changes that occur earlier or later in the progression of ED. A more comprehensive analysis of temporal changes, based on patterns of expression over time, would also be helpful in delineating those genes that might directly lead to ED. However, the high costs of evaluating large-scale gene expression (e.g., GeneChips) restricted the design of our present study. Related to this, we were not able to examine gene expression patterns from multiple organs or tissues, which may be a useful strategy to differentiate between global versus tissue-specific expression changes in response to diabetes. By surveying expression patterns across several tissues, Knoll and colleagues (66) were able to identify common and tissue-specific transcriptional changes in response to short-term diabetes in rats. Finally, although GO analysis is reasonably objective in design, we acknowledge that literature mining as it was used here is a rather subjective method to identify genes of interest. Nevertheless, PubMatrix was a useful tool to generate a vascular-focused and disease-relevant annotation of our microarray data. We think that such an approach is appropriate given the vascular nature of erectile tissue and the physiology of penile erection. The availability of raw Affymetrix files in the GEO repository will allow other researchers to apply different data-mining strategies and analysis techniques to our data. Also, future analysis combining data sets from other research groups using microarrays to study ED may be useful to identify common mechanisms present in the different animal models of ED (119).
Summary.
This study expands the scope of potential candidate genes and pathways that are dysregulated by diabetes and that could negatively impact erectile function. On the basis of GO classification, there was an enrichment of dysregulated ECM genes (e.g., collagen and elastin related) that may have important functions related to cavernosal structure (e.g., Lox). Also, we focused on the discovery of novel gene changes that may affect the proper function of vascular cells in the penis (e.g., Cp) and therefore contribute the development of ED. The various genes and molecules that have been identified in our study can be further evaluated as possible diagnostic tools (e.g., biomarkers) or potential drug targets in patients with diabetes-associated ED.
GRANTS
This study was funded by National Institute of Diabetes and Digestive and Kidney Diseases Grants 1RO1-DK-55017, 5T32-DK-007779, and 5U24-DK-058813.
ACKNOWLEDGMENTS
Present address of M. A. Peters: Rosetta Biosoftware, 401 Terry Ave. N., Seattle, WA 98109.
FOOTNOTES
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
Address for reprint requests and other correspondence: H. Wessells, Dept. of Urology, Harborview Medical Center, 325 9th Ave., Box 359868, Seattle, WA 98104-2499 (e-mail: wessells{at}u.washington.edu).
10.1152/physiolgenomics.00112.2005.
1 The Supplemental Material for this article (Supplemental Figs. IIII) is available online at http://physiolgenomics.physiology.org/cgi/content/full/00112.2005/DC1.
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