Hereditary influences in oxygen-induced retinopathy in the rat.

Rodent models of oxygen-induced retinopathy (OIR) provide important insights into the pathogenesis of human retinopathy of prematurity. Herein, we present an overview of our work with rat OIR to date. We have identified marked and consistent variations in susceptibility to OIR amongst different inbred rat strains and provide strong evidence for a genetic determinant of susceptibility to OIR. Furthermore, we have characterised differences in retinal angiogenic factor gene expression amongst different inbred rat strains exposed to cyclic hyperoxia. A key determinant of susceptibility to OIR appears to be the extent to which pro-angiogenic factor genes, such as vascular endothelial growth factor and erythropoietin, are expressed during the period of hyperoxic exposure. Those strains in which expression is relatively well maintained are less susceptible to retinopathy than are those in which expression is reduced. In addition, we identify an association between ocular pigmentation and OIR susceptibility.

Lentivirus-mediated gene transfer of interleukin 10 to the ovine and human cornea.

BACKGROUND: Gene transfer to a donor cornea ex vivo can modulate corneal graft failure in experimental animal models. We compared a lentiviral vector (LV) carrying the transgene ovine interleukin 10 (IL10) with a comparable adenoviral vector (Ad) for its ability to transduce ovine and human corneas and to modulate ovine corneal allograft survival. METHODS: The LV carrying the ovine IL10 gene was used to transduce ovine and human corneas in vitro. LV-mediated gene expression in corneal endothelium was assessed by real-time quantitative reverse-transcriptase polymerase chain reaction, at varying doses and duration of transduction. The effect of ex vivo transduction of the donor cornea with LV-SV40-IL10 was assessed following orthotopic corneal transplantation in outbred sheep. RESULTS: Expression of IL10 mRNA in Ad-CMV-IL10-transduced ovine corneas was 10(3)-fold higher than in LV-SV40-IL10-transduced corneas (P < 0.0001), and 10(7)-fold higher than in non-transduced controls. IL10 was secreted rapidly from Ad-CMV-IL10-transduced, organ-cultured corneas, peaking at 13-15 days. IL10 secreted from LV-SV40-IL10-transduced corneas increased 20-fold compared with controls, but had not reached a plateau at 15 days. Gene expression driven by LV-SV40-IL10 varied with vector dose and transduction time, but was less than with Ad-CMV-IL10 at both mRNA and protein levels. Gene expression driven by LV-SV40-IL10 was faster in the human cornea than the ovine cornea. Corneal allograft survival was prolonged by a median of 7 days in the LV-SV40-IL10-treated recipients, compared with the control group (P = 0.026). CONCLUSION: Although lentiviral vectors show some promise for corneal gene therapy, they are less efficient than adenoviral vectors.

The potential of viral vector-mediated gene transfer to prolong corneal allograft survival.

The cornea is a particularly attractive target for gene therapy designed to improve the outcome of corneal transplantation. First, there is a clear and well-defined clinical need. Second, because donor corneas can be preserved for days if not weeks within an eye bank, ex vivo transduction of a donor cornea can be carried out without the urgency associated with many other forms of transplantation. Finally, the partial sequestration of the eye from the systemic circulation decreases the likelihood of spillover of vector and transgene, and the immune privileged nature of the cornea and anterior segment affords a degree of protection from immune responses directed against the vector. A wide range of vectors has been investigated for gene transfer to the cornea. A number of viral vectors, in particular, have proved to be efficient at transducing the cornea and in association with a variety of transgenes, have been used successfully to prolong corneal allograft survival significantly in animal models. The most suitable such vector for future clinical studies in corneal transplantation has yet to be determined, but the most likely include recombinant adenoviral, adeno-associated viral and lentiviral vectors. In this review, we examine the ability of these viral vectors to transduce the cornea, and summarise those studies in which gene therapy has been used to prolong experimental corneal allograft survival.

PCR amplification of the functional immunoglobulin heavy chain variable gene from a hybridoma in the presence of two aberrant transcripts.

Single chain antibody fragment genes are commonly created by splicing together the immunoglobulin light chain (VL) and heavy chain variable (VH) genes of a monoclonal antibody produced by a hybridoma. Selective PCR amplification of the functional immunoglobulin variable gene rearrangements can be complicated by the existence of other unproductive immunoglobulin gene rearrangements in the hybridoma. Here we report the detection and preferential amplification of aberrant transcripts from two unproductive VH gene rearrangements derived from the fusion partner of a hybridoma. The functional VH gene of the monoclonal antibody was successfully amplified by selective use of primers to individual JH segments.

Genetic influences on susceptibility to oxygen-induced retinopathy.

PURPOSE: To investigate the inheritance of susceptibility to oxygen-induced retinopathy in the rat with the use of formal backcross analysis. METHODS: Neonatal offspring of inbred albino Fischer 344 (F344) and pigmented Dark Agouti (DA) crosses and F1xF344 and F1xDA backcrosses were exposed to alternating 24-hour cycles of hyperoxia (80% oxygen in air) and normoxia (21% oxygen in air) for 14 days. Retinal avascular area was analyzed by staining with Griffonia simplicifolia isolectin B4, a marker of vascular endothelial cells. Expression of erythropoietin (EPO) mRNA in retinas was quantified by real-time reverse-transcription polymerase chain reaction. RESULTS: Oxygen-exposed offspring of two F344xDA F1 crosses showed retinal avascular areas and ocular and coat pigmentation that were similar to those of the DA strain. Mean retinal avascular area was 73%. Offspring of two DAxF1 backcrosses were similar to F344xDA F1 pups, with pigmented eyes and coats and a mean retinal avascular area of 76%. In contrast, offspring of two F344xF1 backcrosses exhibited a range of eye and coat pigmentation. Mean retinal avascular area of pigmented offspring of the F344xF1 backcrosses was 71% (P < 0.001 compared with F344 rats). Mean avascular area of albino offspring of the F344xF1 backcrosses was 27% (P > 0.05 compared with F344 rats). The normalized expression of EPO mRNA was 3.01 +/- 1.00 in retinas from pigmented F344xF1 backcross offspring compared with 1.31 +/- 0.69 for albino offspring (P < 0.001). CONCLUSIONS: Segregation of the susceptibility trait to oxygen-induced retinopathy in the DA and F344 rat strains is associated with pigmentation and erythropoietin expression and can be modeled using an autosomal dominant pattern of inheritance.

Single chain antibody fragments for ocular use produced at high levels in a commercial wheat variety.

We are investigating the use of single chain antibody fragments (scFv) in eye drops for diagnosis and treatment of eye diseases. For ocular use, recombinant proteins must be free of bacterial endotoxin that causes inflammation in the eye. We required a means of generating high yields of scFvs with little endotoxin contamination. Using microprojectile bombardment we produced transgenic lines of the commercial wheat variety, Westonia, that express two scFvs that bind to CD4 or CD28 on the surface of rat thymocytes. A high level of expression of active scFv in the range 50-180 microg/g was measured by quantitative flow cytometry in crude extracts made from mature seeds. The levels of expression were stable over four generations of transgenic plants and mature seeds were stored for one year with little loss of scFv activity. Substantial purification of scFv was achieved by immobilised metal affinity chromatography. Compared to bacterial extracts, crude transgenic seed extracts contained only a small amount of endotoxin (150 EU/ml) that will be easily removed by purification. The transgenic wheat lines express functional scFv at levels comparable to production in bacteria and promise to be superior to bacteria for production of scFv pharmaceuticals for ocular use.

Interleukin-10 Gene Transfer in Rat Limbal Transplantation.

PURPOSE: To evaluate the gene transfer of the interleukin (IL)-10 cytokine as a treatment modality for prolonging limbal allograft survival in a rat model. MATERIALS AND METHODS: Adenoviral (AV) and lentiviral (LV) vectors were produced for ex vivo gene transfer into limbal graft tissue prior to orthotopic transplantation. Experimental groups comprised unmodified isografts, unmodified allografts, allografts transfected with a reporter gene, and allografts transfected with IL-10. The functional effects of the transgenes were determined by clinical assessment and by following donor cell survival in the recipient animal. Group comparisons were made using survival analysis and tested with the log-rank test. Differences in mean rejection times between groups were tested using the Wilcoxon rank-sum test. RESULTS: Isografts survived during the entire observation period of 56 days. Allografts underwent clinical rejection at a mean of 6.7 days (standard deviation 2.0) postoperatively, irrespective of the presence of transgenes (p < 0.001 for difference in rejection times). For both the AV and LV vector systems, Kaplan-Meier analysis showed a statistically significant difference with respect to time-to-graft failure when comparing allografts transfected with IL-10 with allografts transfected with reporter gene alone (p = 0.011 and p < 0.001, respectively). In the isografts, donor cells could be detected during the complete observation period. In all the allograft groups, however, donor cell detection declined after 1 week and was lost after 4 weeks. CONCLUSIONS: Under the conditions tested in the present model, both the AV and the LV vector systems were able to transfect limbal graft tissue ex vivo with biologically active IL-10, leading to delayed rejection compared to the controls.

Strain-dependent differences in oxygen-induced retinopathy in the inbred rat.

PURPOSE: To examine the susceptibilities of different rat strains to oxygen-induced retinopathy, a model of human retinopathy of prematurity. METHODS: Litters of newborn rats of five inbred strains (Fischer 344 [F344], Dark Agouti [DA], Sprague-Dawley [SD], Wistar-Furth [WF], Lewis [LEW]) and one outbred strain (Hooded Wistar [HW]) were maintained in room air or were exposed to alternating 24-hour cycles of hyperoxia (80% oxygen in air) and normoxia (21% oxygen in air) for 14 days and were killed for analysis, either immediately (postnatal day 14, [P14]) or after 4 days in room air (P18). The fluorophore-conjugated isolectin GS-IB4 was used to label the endothelial cells of wholemounted retinas, and digital images were analyzed for avascular area and for morphologic abnormalities. RESULTS: Exposure to cyclic hyperoxia inhibited retinal vascularization in all strains relative to age-matched room air control animals. Total retinal avascular area at P14 after cyclic hyperoxia varied significantly among strains (P < 0.001). Avascular areas were smallest for the albino F344, WF, and LEW strains; larger for the albino SD strain; and largest for the pigmented DA and HW strains. Susceptibility to hyperoxic vascular attenuation was associated with ocular pigmentation, but neither with body mass nor with natural variation in litter size. Room air exposure for 4 days after cyclic hyperoxia was also associated with strain-related differences in retinal vascularization and with abnormalities in vascular morphology (P < 0.05). For all strains, the size of the avascular retinal area at P14 was predictive of the severity of morphologic abnormality at P18. CONCLUSIONS: Marked and consistent variations in the response of different inbred rat strains to cyclic hyperoxia were observed, suggestive of a genetic component to oxygen-induced retinopathy.

Local gene transfer to modulate rat corneal allograft rejection.

PURPOSE: Allograft rejection is the leading cause of corneal graft failure. CD4(+) T cells control the allograft response and represent targets for antirejection therapy. The purpose of this study was to transfer cDNA encoding a monomeric anti-CD4 antibody fragment to donor corneal endothelium, to attempt to modulate orthotopic corneal allograft rejection in the rat. METHODS: A replication-deficient adenoviral vector (AdV) encoding anti-CD4 single-chain, variable-domain antibody fragment (scFv) and enhanced green fluorescent protein (eGFP) was constructed (AdCD4GFP). AdV encoding eGFP alone (AdGFP) was used as a control. Transgenic product was detected by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, flow cytometry, and fluorescence microscopy. The alloinhibitory capacity of anti-rat CD4 scFv was measured in the one-way mixed lymphocyte reaction (MLR). The survival of Wistar-Furth corneas transduced with AdV either immediately or 3 days before orthotopic transplantation in Fischer 344 recipients was examined. RESULTS: ScFv and eGFP mRNAs were detected in rat corneas transduced in vitro, and active scFv secreted in corneal supernatants peaked at days 4 to 5 after transduction at 23 +/- 4 ng of protein per cornea per day. Antibody and scFv against rat CD4 blocked alloproliferation in MLR. However, transduction of corneas with AdCD4GFP ex vivo, immediately before transplantation, or in vivo, 3 days before transplantation, did not significantly prolong corneal allograft survival (P > 0.05). CONCLUSIONS: Anti-CD4 scFvs were capable of blocking allostimulation, but their local expression within the eye did not prolong corneal allograft survival, suggesting that sensitization may still occur.

Antigens of selected Acanthamoeba species detected with monoclonal antibodies.

Acanthamoeba species are ubiquitous soil and freshwater protozoa that have been associated with infections of the human brain, skin, lungs and eyes. Our aim was to develop specific antibodies to aid in rapid and specific diagnosis of clinically important isolates. Mice were variously immunised with live mixtures of Acanthamoeba castellanii strain 112 (AC112) trophozoites and cysts, or with sonicated, formalin-fixed or heat-treated trophozoites, or with a trophozoite membrane preparation. Eight hybridoma cell lines secreting monoclonal antibodies reactive with A. castellanii epitopes were generated. Seven of the new antibodies (designated AMEC1-3 and MTAC1-4) were isotyped as IgMkappa and one (MTAC5) as IgG1kappa. All of the novel antibodies bound to AC112 cysts, and MTAC4 and MTAC5 also bound to trophozoites as measured by flow cytometry on unfixed cells. Single chain antibody fragments that retained parental antibody binding characteristics were engineered from three of the hybridomas (AMEC1, MTAC3 and MTAC4). Four monoclonal antibodies (AMEC1, AMEC3, MTAC1, MTAC3) bound reliably to unfixed cysts of clinical isolates of A. castellanii (two strains) and Acanthamoeba polyphaga (two strains), belonging to Pussard-Pons morphological group II, and to Acanthamoeba lenticulata and Acanthamoeba culbertsoni, belonging to Pussard-Pons morphological group III. None of the antibodies bound to cysts or trophozoites of the environmental group I species, Acanthamoeba tubiashi. Antibodies AMEC1, MTAC3, MTAC4 and MTAC5 reacted with buffered formalin-fixed AC112 by immunohistochemistry, and also stained Acanthamoeba in sections of infected rat cornea and buffered formalin-fixed, paraffin-embedded infected human cornea. These antibodies may be useful in diagnosing pathogenic Acanthamoeba species in clinical specimens, provided that cysts are present.

Expression of Drosophila Ca2+ permeable transient receptor potential-like channel protein in a prostate cancer cell line decreases cell survival.

The effects of expression of Drosophila melanoga ster Ca(2+) permeable transient receptor potential-like (TRPL) channels, under the control of the cytomegalovirus (CMV) or prostate cell-specific promoters, on cell survival and apoptosis in the androgen-sensitive LNCaP prostate cancer cell line were investigated. A prostate-specific antigen (PSA) promoter construct (designated PSAEn/PSAPr) composed of a 0.6 kb region of the promoter and a 1.45 kb region of the enhancer resulted in androgen-dependent and prostate-specific expression of a luciferase reporter gene in transiently transfected LNCaP cells. Expression of the enhanced green fluorescence protein-TRPL chimeric protein under the control of the CMV promoter was confirmed by Western blot. Whereas the majority of the expressed protein was located in the cytoplasmic space, confocal microscopy with the CD-9 protein as a plasma membrane marker demonstrated that approximately 10% of the expressed TRPL protein was located in a band in the plasma membrane. Using recombinant adenoviruses, expression of the TRPL protein was associated with an increase in both the initial and sustained rates of Ca(2+) inflow. Expression of TRPL under the control of the CMV promoter for 96 hours decreased cell number and increased the number of cells undergoing apoptosis by 23 and 27%, respectively. Apoptosis was inhibited by a caspase-3 inhibitor, Z-DEVD-fmk. It is concluded that, when heterologously expressed in LNCaP cells, the TRPL protein leads to a reduction in cell survival due, in part, to the induction of apoptosis. The effects of TRPL are likely caused by enhanced Na(+) and Ca(2+) inflow to the cells. This finding suggests a novel approach to modify the growth of prostate cancer cells that fail to undergo apoptosis following androgen ablation therapy.

Co-expression of a scFv antibody fragment and a reporter protein using lentiviral shuttle plasmid containing a self-processing furin-2A sequence.

It is often desirable to co-express a reporter protein with a potential therapeutic protein, to verify correct targeting of an expression strategy. Vectors containing a viral self-processing 2A sequence have been reported to drive equimolar expression of two or more transgenes from a single promoter. Here, we report on the co-expression of a secreted antibody fragment and an intracellular reporter protein, enhanced yellow fluorescent protein from lentiviral shuttle plasmids by inserting a furin-2A (F2A) sequence between the two cDNAs, in two different orientations, in the expression cassette. We show that the order of these two transgenes relative to the F2A sequence affects expression levels. Reduced expression of each transgene positioned downstream of F2A, compared with upstream of F2A, was observed (p<0.05). Moreover, protein expression from double-cDNA plasmids was significantly lower than from their corresponding single transgene counterparts (p<0.05).

Expression of an anti-CD4 single-chain antibody fragment from the donor cornea can prolong corneal allograft survival in inbred rats.

AIM: To investigate whether expression of an anti-CD4 antibody fragment (scFv) by a lentivector-transduced donor cornea can prolong rat corneal allograft survival. METHODS: Inbred Fischer 344 rats received penetrating corneal allografts from Wistar-Furth donors after a 3 h transduction of the donor cornea with a lentivector carrying anti-CD4scFv cDNA (Lv-CD4scFv), a lentivector carrying the reporter gene-enhanced yellow fluorescence protein (LV-eYFP), or an adenoviral vector carrying anti-CD4 scFv cDNA (Ad-CD4scFv). Unmodified controls were also performed. Graft survival was assessed by corneal clarity, and rejection was confirmed histologically. RESULTS: In organ-cultured corneas, expression of anti-CD4 scFv was detected at 2 days post-transduction with the adenoviral vector, compared with 5 days post-transduction with the lentivector, and was 10-fold higher than the former. More inflammation was observed in Ad-CD4scFv-modified allografts than in Lv-CD4scFv-modified grafts at 15 days postsurgery (p=0.01). The median time to rejection for unmodified, LV-eYFP and Ad-CD4scFv grafts was day 17, compared with day 22 for Lv-CD4scFv grafts (p≤0.018). CONCLUSION: Donor corneas transduced with a lentiviral vector carrying anti-CD4scFv cDNA showed a modest but significant prolongation in graft survival compared with unmodified, Lv-eYFP and Ad-CD4scFv grafts. However, rejection still occurred in all Lv-CD4scFv grafts, indicating that sensitisation may have been delayed but was not prevented.

The influence of cervical and thoracic lymphadenectomy on corneal allograft rejection in inbred rats.

AIM: To investigate the site of alloantigen presentation in the rat following orthotopic corneal transplantation. METHODS: Adult inbred Fischer 344 rats received penetrating corneal allografts from inbred Wistar Furth donors (n=17), without lymphadenectomy. A second group (n=8) underwent bilateral removal of superficial cervical and facial lymph nodes 7 days before transplantation. A third group (n=9) underwent bilateral removal of superficial cervical, facial, internal jugular and posterior cervical nodes. Graft survival was assessed by corneal clarity and rejection was confirmed histologically. RESULTS: All allografts underwent rejection. The median time to rejection for unmodified allografts was day 15, compared with day 14.5 for minimally lymphadenectomised recipients and day 18 for more extensively lymphadenectomised recipients (p>0.05, all comparisons). The median day to rejection for the combined group of lymphadenectomised rats was day 17 (p>0.05 compared with unmodified grafts). The rejection process was similar in all recipients. CONCLUSIONS: Removal of multiple lymph nodes in the neck and thorax did not significantly influence the incidence, tempo or nature of the corneal allograft response. Sensitisation and clonal expansion of corneal alloantigen-reactive cells cannot occur only in superficial cervical, facial, internal jugular and posterior cervical lymph nodes in the rat.

Production of scFv antibody fragments from a hybridoma with functional activity against human vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) plays a major role in the development of aberrant neovascularization in ocular diseases such as diabetic retinopathy and age-related macular degeneration (ARMD), and is an important therapeutic target for these diseases. Monoclonal antibodies specific for VEGF are in clinical use for some patients with ARMD, delivered by intraocular injection. We have shown previously that single chain antibody fragments (scFv) penetrate into the eye when applied topically to the ocular surface. Here we describe the production of a scFv from a monoclonal antibody specific for human VEGF and demonstrate its ability to decrease proliferation of human umbilical vein endothelial cells in culture. A suitably formulated anti-VEGF scFv may have potential as a less invasive topical treatment for potentially blinding neovascular diseases of the eye.

Gene expression microarray analysis of early oxygen-induced retinopathy in the rat.

Different inbred strains of rat differ in their susceptibility to oxygen-induced retinopathy (OIR), an animal model of human retinopathy of prematurity. We examined gene expression in Sprague-Dawley (susceptible) and Fischer 344 (resistant) neonatal rats after 3 days exposure to cyclic hyperoxia or room air, using Affymetrix rat Genearrays. False discovery rate analysis was used to identify differentially regulated genes. Such genes were then ranked by fold change and submitted to the online database, DAVID. The Sprague-Dawley list returned the term "response to hypoxia," absent from the Fischer 344 output. Manual analysis indicated that many genes known to be upregulated by hypoxia-inducible factor-1alpha were downregulated by cyclic hyperoxia. Quantitative real-time RT-PCR analysis of Egln3, Bnip3, Slc16a3, and Hk2 confirmed the microarray results. We conclude that combined methodologies are required for adequate dissection of the pathophysiology of strain susceptibility to OIR in the rat. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12177-009-9041-7) contains supplementary material, which is available to authorized users.

Kinetics of strain-dependent differential gene expression in oxygen-induced retinopathy in the rat.

Recent evidence suggests that retinopathy of prematurity, a potentially blinding condition of premature human neonates, has a genetically-determined component. Different inbred strains of rat exhibit differential susceptibility to oxygen-induced retinopathy (OIR), a well-established experimental model of retinopathy of prematurity. To explore the basis for this differential susceptibility, we quantified the retinal expression of 8 angiogenesis-related genes during early post-natal retinal development in rats with OIR. Inbred Fischer 344 (F344), Dark Agouti (DA) and Sprague Dawley (SPD) rat neonates were exposed to alternating cycles of 80% oxygen in air and normoxia for up to 14 days. After 14 days of cyclic hyperoxic exposure, some rats were exposed to normoxia for a further 4 days. Retinal mRNA for vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), pigment epithelium-derived factor (PEDF), angiopoietin-2 (Ang2), Tie2, cyclooxygenase-2 (COX2), insulin-like growth factor-1 (IGF1) and erythropoietin (EPO) were quantified by real-time reverse-transcriptase polymerase chain reaction at different time-points. Time-course analysis showed that expression of mRNA for VEGF, VEGFR2 and Ang2 was significantly greater in OIR-resistant (F344) retinae than in OIR-susceptible (DA) retinae during the first 9 days of cyclic hyperoxia. However, at post-natal days 14 and 18, retinal mRNAs for VEGF, EPO, VEGFR2, Ang2, IGF1, COX2 and PEDF were expressed to a significantly greater extent in OIR-susceptible (DA, SPD) than OIR-resistant (F344) retinae. The VEGF/PEDF ratio was greater in the F344 compared with the DA strain up to day 9, but was higher in the DA than the F344 strain at days 14 and 18. Thus, we found that retinal expression of angiogenesis-related genes was significantly higher in OIR-resistant rats than in OIR-susceptible rats during early retinal development, but the pattern reversed during the proliferative phase of OIR. We conclude that susceptibility to OIR correlates with differential gene expression very early in retinal microvascular development, during periods of cyclic hyperoxic exposure rather than during subsequent sustained hypoxia.

Evidence for the expression of transient receptor potential proteins in guinea pig airway smooth muscle cells.

OBJECTIVE: The present study investigates the expression of transient receptor potential (TRPC) proteins in airway smooth muscle (ASM) cells in order to determine whether these proteins may be candidate molecular counterparts of plasma membrane Ca2+-permeable channels involved in the contraction of ASM. METHODS: Expression of TRPC mRNA was detected using specific primers and RT-PCR. Expression of the TRPC1, TRPC3 and TRPC6 proteins was detected using antibodies in immunoprecipitation and Western blot. RESULTS: Guinea pig ASM cells exhibited thapsigargin- and acetylcholine-initiated Ca2+ inflow but none by 1-oleoyl-2-acetyl-sn-glycerol. mRNA encoding each of the TRPC1 to TRPC6 proteins was detected in ASM cells. mRNA encoding TRPC1, TRPC3, TRPC4 and TRPC6 was detected in ASM cells at a concentration approximately equivalent to that in guinea pig brain. mRNA encoding TRPC2 and TRPC5 was more abundant in ASM cells than in brain. The TRPC1 protein, but not the TRPC3 or TRPC6 proteins, was detected in extracts of ASM cells, while all three proteins were detected in brain. CONCLUSION: The results provide evidence for a low level of expression of the TRPC1 to TRPC6 proteins in ASM cells. These proteins may function as store-operated Ca2+ and/or second messenger-activated non-selective cation channels in ASM cells.