|Molecular Vision 2004;
Received 22 July 2003 | Accepted 16 December 2003 | Published 17 February 2004
Expression pattern of alternatively spliced PECAM-1 isoforms in retinal vasculature
Yongji Wang,1 Kristin Repyak,2
Departments of 1Ophthalmology & Visual Sciences, 2Pediatrics, and 3Pharmacology, University of Wisconsin Medical School, Madison, WI
Correspondence to: Nader Sheibani, Department of Ophthalmology & Visual Sciences, University of Wisconsin Medical School, 600 Highland Avenue, K6/458 CSC, Madison, WI, 53792-4673; Phone: (608) 263-3345; FAX: (608) 265-6021; email: firstname.lastname@example.org
Purpose: Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is a cell adhesion-signaling molecule with important roles in angiogenesis and inflammation. The alternative splicing of the PECAM-1 cytoplasmic domain modulates its adhesive properties during vascular development and angiogenesis. This study was designed to identify alternatively spliced PECAM-1 isoforms in human and murine retina and during postnatal vascularization of murine retina.
Methods: RT-PCR, DNA sequencing, and Western blot analysis were utilized to examine the expression pattern of alternatively spliced PECAM-1 isoforms in human and mouse retina, and during vascularization of murine retina.
Results: We demonstrate that the PECAM-1 cytoplasmic domain undergoes alternative splicing generating multiple isoforms in vascular beds of human and mouse retina. We detected full length PECAM-1 and an isoform that lacks exon 14 (Δ14) in human retina. Seven isoforms of PECAM-1 were detected in murine retina. These included full length, Δ12, Δ14, Δ15, Δ12&15, Δ14&15, and Δ12, 14&15 PECAM-1 isoforms. The full length PECAM-1 was the predominant isoform detected in human retina, while the isoform lacking exon 14&15 (Δ14&15) was the predominant isoform detected in murine retina. In addition, the expression pattern of PECAM-1 isoforms changed during vascularization of murine retina.
Conclusions: PECAM-1 mRNA undergoes alternative splicing generating multiple isoforms in human and murine retinal vasculature. The regulated expression pattern of these isoforms may influence endothelial cell adhesive properties impacting vasculogenesis and angiogenesis.
PECAM-1 (CD31) is a member of the immunoglobulin gene superfamily that is highly expressed on the surface of endothelial cells and to a lesser extent on hematopoietic cells. PECAM-1 is encoded by a relatively large gene (110 Kbp) containing 16 exons encoding the 6 Ig-like extracellular domains (exons 3-8), a transmembrane domain (exon 9), and a relatively long cytoplasmic domain (exons 10-16) [1,2]. Exons 1 and 2 encode 5'-untranslated region and the signal peptide. The cytoplasmic domain of PECAM-1 (exons 10-16) undergoes alternative splicing generating multiple isoforms, whose expression is developmentally regulated .
PECAM-1 participates in both homophilic [4-6] and heterophilic [7-10] interactions influencing cell adhesive mechanisms. The PECAM-1 cytoplasmic domain plays an active role in modulation of cellular adhesive properties. The lack of exon 14 and/or its tyrosine phosphorylation is shown to modulate PECAM-1 homophilic and heterophilic adhesive properties when expressed in L-cells [11,12]. We have recently demonstrated that expression of different PECAM-1 isoforms, with and without exon 14, in MDCK (Madin-Darby canine kidney) cells differentially modulate their ability to form cadherin-mediated cell-cell adhesion . This is mediated by the differential ability of the PECAM-1 isoforms to activate MAPK/ERKs pathway. These cells, unlike L-cells, form cadherin-mediated cell-cell interactions very similar to endothelial cells but lack PECAM-1 expression. Therefore, PECAM-1 isoforms play an active role in regulation of cell adhesive properties through their specific interactions with intracellular signaling proteins.
PECAM-1 mediated cell-cell interactions are important during angiogenesis and inflammation . PECAM-1 deficient mice appear to develop normally, but exhibit a number of vascular defects during inflammation. The diapedesis of leukocytes is significantly delayed in PECAM-1 deficient mice concomitant with persistent permeability defects, as well as defects in neovascularization of the granulation tissue, during inflammatory challenges [15,16].
Angiogenesis, the formation of new blood vessels from pre-existing capillaries, and vasculogenesis, the differentiation and organization of precursor endothelial cells into a vascular network, are both reported to mediate retinal vascularization [17-24]. The murine retinal vasculature develops postnatally and provides a unique opportunity to study all aspects of vascular development, remodeling, and maturation. Retinal vasculature is restricted to two dimensions, simplifying the study of a vascular plexus in its entirety. The development of retinal vasculature has been the focus of a large body of literature, in particular in the context of retinopathies in which abnormal vessel growth in the retina can ultimately lead to blindness. However, the development of this vasculature at the molecular and cellular levels requires further delineation. Recent studies on the function of different vascular endothelial growth factor (VEGF) isoforms, an important angiogenic factor, in development of murine retinal vasculature indicated that VEGF isoforms are selectively expressed with distinct roles in retinal vascular patterning and arterial venual development .
The expression and distribution of PECAM-1 isoforms in retinal vasculature have not been previously examined. Our recent studies indicate that the expression pattern of PECAM-1 isoforms is developmentally regulated and these isoforms can differentially modulate cell adhesive properties [3,13]. Therefore, a better understanding of how the expression of PECAM-1 isoforms relates to the development of retinal vasculature may provide further insight into the function of PECAM-1 in vascular development and angiogenesis.
Here, we demonstrate that multiple isoforms of PECAM-1 are expressed in vascular beds of human and murine retina. The full length PECAM-1 is the predominant isoform detected in vascular beds of human retina. This is in contrast to murine retina, where the isoform that lacks exons 14 and 15 is the predominant isoform. In addition, the expression pattern of PECAM-1 isoforms changed during vascularization of retina. Thus, the exonic inclusion and/or exclusion may play a role during vascularization of retina by modulating PECAM-1 mediated endothelial cell adhesive properties.
The mouse brain endothelial (bEND) cells have been previously described  and maintained in Dulbecco's modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C and 10% CO2. The MDCK cells expressing vector Δ14&15 or Δ15 murine PECAM-1 isoforms were generated as previously described  and maintained in minimum essential medium supplemented with 10% FBS, 25 mM HEPES, and 400 μg/ml of G418 at 37 °C and 5% CO2. The human 293 cells were obtained from ATCC (Manassas, VA) and maintained in DMEM supplemented with 5% FBS at 37 °C and 5% CO2.
Human retinas were obtained from postmortem donor eyes (Wisconsin Lions Eye Bank, Madison, WI) within 24 h of death. Murine retinas were obtained from 3-days to 6-weeks of age FVBN mice (Harlan, Indianapolis, IN). The eyes were enucleated, hemisected, lens and vitreous were removed, and retinas were dissected out under a dissecting microscope in cold phosphate buffered saline (PBS). The retinas were washed with DEPC-treated PBS for RNA isolation, or with cold PBS for preparation of retina extracts for Western blot analysis. All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Total RNA isolation
Total RNA isolation was performed using the single step RNA isolation (RNAwizTM, Ambion, Austin, TX). Retinas from a single human eye or 4-6 mice were homogenized in a 0.5 ml of RNAwiz solution. Total RNA was prepared as recommended by the supplier, and stored at 80 °C until needed.
RT-PCR analysis and DNA sequencing
Total RNA was utilized as template for RT-PCR (SuperscriptTM One-Step RT-PCR, Invitrogen, Carlsbad, CA) to amplify the cytoplasmic domain of all possible PECAM-1 isoforms. The sense primers were designed as 5'-atggatcc 2021AGG AAA GCC AAG GCC AGG2038-3' for human PECAM-1, and 5'-atggatcc1941AGG AAA GCC AAG GCC AAA1958-3' for murine PECAM-1, which spans the border of exon 9 and exon 10 within the cytoplasmic domain. The anti-sense primers were designed as 5'-cggaattc 2371CCT TGC TGT CTA AGT CCT2354-3 for human PECAM-1, and 5'-cggaattc 2291TTG ACT GTC TTA AGT TCC2274-3' for murine PECAM-1, which spans the border of exon 16 and 3'-untranslated region. The primers carry a BamHI and an EcoRI recognition sequence (lowercase letters) to facilitate subsequent cloning. The PCR products were examined on 2.4% agarose gels to assess their integrity and expected size. For cloning, PCR products were purified using Spin Q columns (Qiagen, Valencia, CA), digested with BamHI and EcoRI, ligated into the pGEX-2T vector (Amersham, Piscataway, NJ) cut with the same enzymes, and transformed into E. coli DH5α. Bacterial colonies were screened by BamHI and EcoRI digestion of minipreps and those with inserts were sequenced using the Big Dye reagent (Perkin Elmer) as described previously . All PCR reactions were performed in an Eppendorf Gradient Cycler (Westbury, NY). For DNA sequencing the following PCR parameters were used, 96 °C 5 min, followed by 25 cycles of 96 °C for 30 s, 50 °C for 15 s, and 60 °C for 4 min. The DNA samples were ethanol precipitated and prepared for analysis by the DNA sequencing facility at University of Wisconsin Biotechnology Center.
Identification of PECAM-1 isoforms
The exonic mutation sites of PECAM-1 cDNA molecules were identified by comparison of the mutant sequences with that of the wild type [27,28]. The isoform (Δ12) with a new junction in cDNA sequence, that lacks exon 12 was identified at G2135-A2190 (loss of 55 bp); the isoform (Δ14) that lacks exon 14 at G2252-A2310 (loss of 58 bp); the isoform (Δ15) that lacks exon 15 at G2309-A2333 (loss of 24 bp); the isoform (Δ12&15) that lacks exon 12 and 15 at G2135-A2190 and G2309-A2333 (loss of 79 bp); the isoform (Δ14&15) that lacks exon 14 and 15 at G2252-A2333 (loss of 81 bp); and the isoform (Δ12,14&15) that lacks exon 12, 14 and 15 at G2135-A2190 and G2252-A2333 (loss of 136 bp). The expected size for the wild type form (full length PECAM-1 cytoplasmic domain) is 351 bp according to the human PECAM-1 cDNA sequence. However, the alternatively spliced isoforms have variable sizes, smaller than the wild type. Absence of exon 15 in Δ15, Δ12&15 and Δ14&15 isoforms changes the reading frame terminating up-stream of the commonly utilized termination codon.
Preparation of antibodies to mouse PECAM-1
The majority of the commercially available PECAM-1 antibodies do not react with the mouse PECAM-1 on Western blots. Therefore, we have developed two polyclonal antibodies, one to the extracellular domain of PECAM-1 which reacts with all isoforms and one to exon 14 of PECAM-1 which reacts with isoforms containing exon 14, as outline here. The cDNA corresponding to the extracellular domain of murine PECAM-1 was generated by PCR using gene specific primers and full-length murine PECAM-1 cDNA. The sense primer was 5'-ccc aag ctt GGG ATG CTC CTG GCT CTG GGA CTC ACG CTG-3' with HindIII site, and the antisense primer 5'-tgc tct aga GCA CCC TTT CTT CCA TGG GGC AAG GAA GAC-3' with Xba I site. The PCR product was purified, digested with HindIII and XbaI, and was inserted into pcDNA3.1MycHisA(+) (Invitrogen, Carlsbad, CA) expression vector cut with the same enzymes. The PECAM-1 cDNA was in frame with the vector sequence at its 3'-end to allow incorporation of Myc and 6Xhis tag as well as the termination codon. Identity of the positive clones was confirmed by restriction enzyme analysis and DNA sequencing. For antigen preparation, the PECAM-1 extracellular domain expressing plasmid was transfected into 293 cells using Lipofectin® Reagent (Invitrogen, Carlsbad, CA). Following transfection, cells were grown in the presence of G418 (500 μg/ml) and a population of cells expressing soluble PECAM-1 was obtained. Soluble PECAM-1 was purified from the conditioned growth medium using Ni-NTA affinity chromatography (Qiagen, Valencia, CA). The quantity of PECAM-1 was assessed by SDS-PAGE and coomassie staining along with known amount of bovine serum albumin. The required amounts of antigen (PECAM-1 bound to Ni-NTA-agarose) were sent to a commercial vender for preparation of rabbit polyclonal antibody (Coclaco, Reamstown, PA). The antiserum was screened using soluble PECAM-1 bound Ni-NTA absorbed strips (Qiagen, Valencia, CA). The antiserum was also reactive with murine PECAM-1 on blots of endothelial cell lysates. The preparation of an anti-murine exon 14 peptide antibody, which exclusively recognizes murine PECAM-1 isoforms with exon 14, has been previously described [3,29].
Tissue lysates, immunoprecipitation, and western blot analysis
For retina protein extract preparations, retina from 2-4 mice were resuspended in 0.5 ml of lysis buffer (20 mM Tris pH 7.4, 2 mM EDTA, 1% Triton X-100, 1% NP-40, 0.5% deoxycholate) and protease inhibitors cocktail (Roche Biochemicals, Indianapolis, IN) and briefly sonicated. Protein concentrations were determined using the BCA protein assay kit (Pierce, Rockford, IL). Equal amounts of protein were utilized for Western blot analysis.
For PECAM-1 immunoprecipitation, bEND or MDCK cells were gently washed with cold TBS (20 mM Tris, 150 mM NaCl pH 7.4) twice, and lysed in 0.5 ml of lysis buffer (20 mM Tris pH 7.4, 2 mM EDTA, 1% Triton X-100, and protease inhibitors cocktail, Roche Biochemicals, Indianapolis, IN) and briefly sonicated. The supernatants were centrifuged at 14,000 xg at 4 °C. Protein concentrations were determined using the BCA protein assay kit (Pierce, Rockford, IL). Equal amounts of cell lysates (200 μg) were utilized for immunoprecipitation of PECAM-1. Briefly, 30 μl of agarose beads coated with 4 μg of anti-PECAM-1 antibody (MEC13.3, BD Pharmingen, San Diego, CA) were incubated with the cell lysates at 4 °C for 2 h. The beads were washed three times with the lysis buffer containing 1% Triton X-100, resuspended in 1 x SDS sample buffer, boiled, and utilized for Western blot analysis.
For Western blot analysis, the retina lysates (25 μg) or immunoprecipitate eluates were analyzed by SDS-PAGE (4-20% Tris-Glycine Gel, Invitrogen, Carlsbad, CA), and transferred to nitrocellulose. The blot was either incubated with an antibody to the PECAM-1 extracellular domain (described above, which recognizes all PECAM-1 isoforms) or the anti-exon 14 antibody, which recognizes isoforms with exon 14. Following incubation with appropriate secondary antibody, blots were washed and developed using ECL (Amersham, Piscataway, NY).
The distribution of PECAM-1 isoforms in human and murine retinal vasculature
The species- and tissue-specific distribution of PECAM-1 isoforms have been recently defined in various tissues and endothelial cells [3,27,28]. However, the expression and distribution of different PECAM-1 isoforms in the retinal vasculature have not been previously reported. The postnatal developing murine retinal vasculature provides a unique opportunity to study the role of PECAM-1 isoforms in all aspects of vascular development, remodeling, and maturation. To determine the expression pattern of PECAM-1 isoforms in human and murine retina, RT-PCR was performed on RNA isolated from human and mouse retinas as described in Materials and Methods. The primers employed encompassed the entire cytoplasmic domain thus having the potential to amplify all PECAM-1 isoforms. The band corresponding to 350 bp is the full-length PECAM-1 cytoplasmic domain. Those band(s) smaller than 350 bp would result from alternative splicing. The RNA samples from human retinal tissue exhibited a RT-PCR pattern with a predominant band corresponding to the expected size of the full-length PECAM-1 cytoplasmic domain (Figure 1). In contrast, multiple banding patterns were observed in murine retinas, suggesting multiple isoforms of PECAM-1 were expressed (Figure 1). Therefore, fewer isoforms of PECAM-1 may be expressed in human retina compared to murine retina. However, it is difficult to resolve all the potential isoforms in this manner. To confirm that these results were due to fewer PECAM-1 isoforms in human retina we next cloned and sequenced the cDNA products generated by RT-PCR.
Identification of PECAM-1 isoforms in human and murine retinal vasculature
The identity of the cDNAs generated by RT-PCR was determined by direct cloning and sequencing as described in Methods. Table 1 shows the isoforms of PECAM-1 and the frequency at which they were detected in human and murine retina. Two isoforms of human PECAM-1 were detected in vascular beds of human retina (Table 1). We detected full length PECAM-1 (93%) as well as the isoform lacking exon 14 (7%). This is consistent with our previous observations in vascular beds of other human tissues, platelets, endothelial, and hematopoietic cells [27,28]. The PECAM-1 isoforms detected in murine retina from P28 mice included the full length PECAM-1 and isoforms lacking exon 12, 14, 15, 14&15, or 12, 14&15. The PECAM-1 Δ14&15 (68%) is the predominant isoform detected in vascular beds of murine retina. The cDNA and amino acid sequences of all the PECAM-1 isoforms detected in this study are shown in Figure 2 and Figure 3, respectively. The absence of exon 15 changes the reading frame resulting in utilization of an upstream termination codon shortening the cDNA by three amino acids and incorporation of six different amino acids upstream of the termination codon (Figure 2 and Figure 3). The putative splice sites of human and murine PECAM-1 mRNA molecules were as previously described [3,27,28].
Modulation of PECAM-1 expression and alternative splicing during vascularization of murine retina
Our previous studies indicated that PECAM-1 isoforms have different adhesive properties and their switching during angiogenesis may be essential for proper formation of blood vessels and their function [3,13]. The studies described above indicated that multiple isoforms of PECAM-1 could be detected in the retina vasculature. To further gain a better understanding of the function of PECAM-1 isoforms during postnatal vascularization of retina, we examined the distribution of PECAM-1 isoforms in retinas from postnatal days 3 to 28 (P3 to P28) mice. The RNA samples prepared from retinas of different aged mice exhibited a similar RT-PCR pattern with multiple bands corresponding to the size of the different PECAM-1 isoforms cytoplasmic domain (Figure 1 and Figure 4). These data suggested that different isoforms of PECAM-1 are expressed during vascularization of murine retina. However, the differences in the intensities of bands detected suggest that stage specific expression of different isoforms during vascularization of retina may exist. To identify these PECAM-1 isoforms in murine retinal vasculature, we next cloned and sequenced the cDNA products generated by RT-PCR.
Distribution of PECAM-1 isoforms during vascularization of murine retina
The identity of the cDNAs generated by RT-PCR was determined by direct cloning and sequencing as described in Materials and Methods. Table 2 shows the isoforms of PECAM-1 and the frequency at which they were detected in vasculature of murine retina. Seven isoforms of PECAM-1 were detected (Table 2). These included full length PECAM-1, and isoforms lacking exon 12, 14, 15, 12& 15, 14&15, or 12, 14&15 (Figure 2 and Figure 3). The PECAM-1 isoform that lacks exon 14 and 15 (Δ14&15) is the predominant isoform detected in vascular beds of murine retinas at all time points. However, changes in the frequencies at which PECAM-1 isoforms were detected were observed during postnatal vascularization of retina. Thus, suggesting that expression of alternatively spliced PECAM-1 isoforms may be regulated during retinal vascularization. We consistently observed a higher frequency of the isoform Δ14&15 during early (P3, P7, and P10) and later (P20 and P28) stages of retinal vascularization, but a lower frequency during the mid-stages when active vascular sprouting of deep retina occurs (P15). The expression of isoform Δ15 increases in frequency with retinal vasculature maturation. Isoform Δ12&15 was only detected in P20 mouse retina. The full-length PECAM-1 and isoforms that lack exon 15 or 12, 14&15 were also detected in the retina vasculature.
Exon 14 of PECAM-1 modulates adhesive properties of PECAM-1 isoforms perhaps through its interaction with intracellular proteins. We recently showed expression of the Δ15 PECAM-1 isoform (containing exon 14) in MDCK cells activates MAPK/ERKs, prevents cadherin-mediated cell-cell interactions, and promotes cell migration. However, expression of the Δ14&15 PECAM-1 isoform (lacking exon 14) in MDCK cells fails to activate MAPK/ERKs and does not affect cadherin-mediated cell-cell interactions . Therefore, we next determined the percentage of PECAM-1 molecules that contain exon 14 (Figure 5C). These results demonstrated that the expression of PECAM-1 isoforms without exon 14 occurs in either earlier or later stages of retinal vascularization. In contrast, isoforms with exon 14 are more predominant during the formation of deep vascular plexus (P10-P20) when active sprouting of new vessels from the superficial layer is occurring. These data are consistent with our previous observations during development of kidney vasculature, where isoforms with exon 14 are expressed early during active angiogenesis and later replaced with isoforms without exon 14 . To our knowledge this is the first investigation demonstrating expression of the alternatively spliced PECAM-1 isoforms during vascularization of murine retina.
Expression of the alternatively spliced PECAM-1 isoforms during vascularization of murine retina
To determine whether alternatively spliced isoforms of PECAM-1 mRNA are translated, we examined the expression pattern of PECAM-1 isoforms during vascularization of murine retina using Western blot analysis. We utilized two different antibodies to murine PECAM-1. One antibody was made to the extracellular domain of murine PECAM-1, which recognizes all PECAM-1 isoforms, and another antibody made to exon 14 of murine PECAM-1, which reacts with isoforms containing exon 14. The specificity of these antibodies is demonstrated in Figure 5A. The antibody against extracellular domain of PECAM-1 reacts with PECAM-1 isoforms (Δ15 and Δ14&15) expressed in MDCK cells as well as PECAM-1 positive mouse brain endothelial (bEND) cells but not vector transfected MDCK cells. The majority of PECAM-1 isoforms expressed in bEND cells lack exon 14 . When the same blot was probed with the antibody to exon 14, a band was only detected in MDCK cells expressing Δ15 PECAM-1 but not Δ14&15 isoform. Figure 5B shows a Western blot of retina extracts prepared from mouse retina at different stages of development blotted with the two different PECAM-1 antibodies. The antibody to extracellular domain of PECAM-1 detected a main band at approximately 120-130 kDa (full length PECAM-1 is ~130 kDa, upper panel) in all the samples. When the same blot was incubated with exon 14 specific antibody, a similar banding pattern was detected, except with different intensities (lower panel), suggesting presence of other isoforms lacking exon 14. The retinas from P7, P28, and P42 expressed significantly lower amounts of PECAM-1 containing exon 14. In contrast, P10, P15, and P20 retinas expressed higher amounts of PECAM-1 containing exon 14 (lower panel) compared to other time points. The quantitative assessments of these data was consistent with the pattern of PECAM-1 isoforms detected in Table 2 and the percentages of isoforms containing exon 14 (Figure 5C). Therefore, murine retina not only expresses multiple isoforms of PECAM-1 during vascularization but their product is also present.
Most tissues are vascularized by organization of pre-existing endothelial precursor cells and/or migration and proliferation of endothelial cells from pre-existing capillaries into a capillary network [30,31]. Vascularization of the retina is believed to involve both of these processes. However, the molecular and cellular mechanisms that contribute to the development of this vasculature require further delineation. PECAM-1 is a cell adhesion signaling molecule whose homophilic and heterophilic interactions play important roles in angiogenesis and inflammation. The regulated expression of PECAM-1 isoforms during vascular development in vivo  and tube formation in vitro  suggest that PECAM-1 function can be modulated by alternative splicing of its cytoplasmic domain. Here, we show that (1) human and murine PECAM-1 undergo alternative splicing in the retina vasculature generating a number of isoforms; (2) two isoforms of PECAM-1 were detected in human retina, the full length and Δ14 PECAM-1, while seven isoforms were detected in murine retina, the full-length, Δ12, Δ14, Δ15, Δ12&15, Δ14&15, and Δ12, 14&15; (3) the predominant isoform detected in human retina was full length PECAM-1, while in murine retina it was the Δ14&15 PECAM-1 isoform; and (4) the expression pattern of PECAM-1 isoforms changed during postnatal vascularization of murine retina.
The structural similarity of retinal vasculature between mouse and human has been observed based on immunohistochemical staining of retinal vessels . In general, the mouse retinal vasculature develops similar to that in humans. In both species the first vessels originate at the optic nerve head and spread over the inner surface of the retina, forming a dense network . In the newborn mouse (postnatal day 0, P0), vascular sprouts emerge from a ring-shaped vessel around the optic nerve head. The primary vascular network spreads approximately halfway across the inner surface of the retina by P4 and reaches the periphery approximately 1 week (P7) after birth. After the vascular network has spread across the entire retina, arteries and veins strictly alternate and vascular sprouts start (~P7) to sprout downward, into the inner plexiform layer, where they establish a second vascular network (~P21) parallel to the first [32-34]. It is a widely held view that the primary vascular development across the inner surface of the retina occurs by vasculogenesis, whereas the establishment of the secondary network in the inner plexiform layer occurs by angiogenesis. Our results show that although development of the retinal vasculature may occurs similarly in human and mouse, the PECAM-1 isoforms present are quite different (Tables 1 and 2). This is consistent with our previous observation in human and murine tissues and endothelial cells [3,27]. The vascular beds of murine tissues exhibit a greater number of different PECAM-1 isoforms than human tissues. Therefore, different mechanisms may modulate PECAM-1 adhesive properties in different species.
The expression pattern of alternatively spliced PECAM-1 isoforms is altered during vascularization of retina. We detected seven isoforms of PECAM-1 in murine retina vasculature (Table 2). Although, the PECAM-1 isoform Δ14&15 was the predominant isoform at all the time points examined, changes in the frequency at which the different PECAM-1 isoforms occurred was observed. The frequency of the Δ14&15 isoform was higher during earlier stages (P3, P7, and P10) and the later stages (P20 and P28) of retinal vascularization, and lower during more active stage of angiogenesis (P15) in murine retina. This is consistent with enhanced migratory phenotype of the MDCK cells expressing a PECAM-1 isoform with exon 14 but not a PECAM-1 isoform lacking exon 14 . We showed this was mainly attributed to sustained activation of MAPK/ERKs in MDCK cells expressing the PECAM-1 isoform with exon 14. This resulted in disruption of cadherin-mediated cell-cell adhesion allowing cells to be more migratory. Furthermore, we have recently shown that sustained activation of MAPK/ERKs in endothelial cells similarly results in enhanced migration and disruption of cadherin-mediated cell-cell interactions promoting tube formation on Matrigel . Therefore, PECAM-1 isoform switching in the mouse may provide a mechanism to modulate endothelial cell adhesive properties during vascular development and angiogenesis. The pattern of isoforms observed in fully vascularized retina was consistent with our previous observation in other mature vascularized murine tissues and endothelial cells .
We observed that expression of the PECAM-1 isoforms with and without exon 14 changes during vascularization of murine retina (Figure 5C). At earlier (P3 and P7) or later (P28 and P42) stages of vascular formation, retinal vasculature expressed PECAM-1 isoforms lacking exon 14. In contrast, during the formation of the secondary layer of retinal vasculature, which occurs through extensive sprouting of the superficial layer vessels, PECAM-1 isoforms containing exon 14 (P10-P20) were predominant. Exon 14 of PECAM-1 plays an important role in regulation of PECAM-1 adhesive properties, perhaps through its interaction with intracellular signaling molecules as described above. Therefore, PECAM-1 isoforms play an active role in regulation of endothelial cell adhesive properties. To our knowledge, this is the first investigation showing differential expression of the alternatively spliced PECAM-1 isoforms during vascularization of murine retina.
The modification of the PECAM-1 cytoplasmic domains by alternative splicing may regulate their role during angiogenesis and/or vasculogenesis. A better understanding of how expression of PECAM-1 isoforms relate, both spatially and temporally, to the whole vascular network may provide insight into the role of PECAM-1 isoforms in regulation of retinal vascular development and neovascularization associated with a number of pathological conditions including retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration.
This investigation was supported in part by the grants AR 45599 and EY 13700 from the National Institutes of Health. NS is a recipient of Career Development Award from Research to Prevent Blindness.
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