Corneal Specialists' Confidence in Identifying Causal Organisms of Microbial Keratitis.

PURPOSE: Microbial keratitis (MK) is a potentially blinding corneal disease caused by an array of microbial etiologies. However, the lack of early organism identification is a barrier to optimal care. We investigated clinician confidence in their diagnosis of organism type on initial presentation and the relationship between confidence and presenting features. METHODS: This research presents secondary data analysis of 72 patients from the Automated Quantitative Ulcer Analysis (AQUA) study. Cornea specialists reported their confidence in organism identification. Presenting sample characteristics were recorded including patient demographics, health history, infection morphology, symptoms, and circumstances of infection. The association between confidence and presenting characteristics was investigated with 2-sample t-tests, Wilcoxon tests, and Chi-square or Fisher's exact tests. RESULTS: Clinicians reported being "confident or very confident" in their diagnosis of the causal organism in MK infections for 39 patients (54%) and "not confident" for 33 patients (46%). Confidence was not significantly associated with patient demographics, morphologic features, or symptoms related to MK. MK cases where clinicians reported they were confident, versus not confident in their diagnosis, showed significantly smaller percentages of previous corneal disease (0% versus 15%, p = 0.017), were not seen by an outside provider first (69% versus 94%, p = 0.015), or had no prior labs drawn (8% versus 33%, p = 0.046), and a significantly larger percentage of cases wore contact lenses (54% versus 28%, p = 0.029). CONCLUSION: In almost half of MK cases, cornea specialists reported lack of confidence in identifying the infection type. Confidence was related to ocular history and circumstances of infection but not by observable signs and symptoms or patient demographics. Tools are needed to assist clinicians with early diagnosis of MK infection type to expedite care and healing.

Automatic Classification of Slit-Lamp Photographs by Imaging Illumination.

PURPOSE: The aim of this study was to facilitate deep learning systems in image annotations for diagnosing keratitis type by developing an automated algorithm to classify slit-lamp photographs (SLPs) based on illumination technique. METHODS: SLPs were collected from patients with corneal ulcer at Kellogg Eye Center, Bascom Palmer Eye Institute, and Aravind Eye Care Systems. Illumination techniques were slit beam, diffuse white light, diffuse blue light with fluorescein, and sclerotic scatter (ScS). Images were manually labeled for illumination and randomly split into training, validation, and testing data sets (70%:15%:15%). Classification algorithms including MobileNetV2, ResNet50, LeNet, AlexNet, multilayer perceptron, and k-nearest neighborhood were trained to distinguish 4 type of illumination techniques. The algorithm performances on the test data set were evaluated with 95% confidence intervals (CIs) for accuracy, F1 score, and area under the receiver operator characteristics curve (AUC-ROC), overall and by class (one-vs-rest). RESULTS: A total of 12,132 images from 409 patients were analyzed, including 41.8% (n = 5069) slit-beam photographs, 21.2% (2571) diffuse white light, 19.5% (2364) diffuse blue light, and 17.5% (2128) ScS. MobileNetV2 achieved the highest overall F1 score of 97.95% (CI, 97.94%-97.97%), AUC-ROC of 99.83% (99.72%-99.9%), and accuracy of 98.98% (98.97%-98.98%). The F1 scores for slit beam, diffuse white light, diffuse blue light, and ScS were 97.82% (97.80%-97.84%), 96.62% (96.58%-96.66%), 99.88% (99.87%-99.89%), and 97.59% (97.55%-97.62%), respectively. Slit beam and ScS were the 2 most frequently misclassified illumination. CONCLUSIONS: MobileNetV2 accurately labeled illumination of SLPs using a large data set of corneal images. Effective, automatic classification of SLPs is key to integrating deep learning systems for clinical decision support into practice workflows.

The effect of social determinants of health on severity of microbial keratitis presentation at a tertiary eye care hospital in Southern India.

Purpose: Understanding the association between social determinants of health (SDoHs) and microbial keratitis (MK) can inform underlying risk for patients and identify risk factors associated with worse disease, such as presenting visual acuity (VA) and time to initial presentation. Methods: This was a cross-sectional study was conducted with patients presenting with MK to the cornea clinic at a tertiary care hospital in Madurai, India. Patient demographics, SDoH survey responses, geographic pollution, and clinical features at presentation were collected. Descriptive statistics, univariate analysis, multi-variable linear regression models, and Poisson regression models were utilized. Results: There were 51 patients evaluated. The mean age was 51.2 years (SD = 13.3); 33.3% were female and 55% did not visit a vision center (VC) prior to presenting to the clinic. The median presenting logarithm of the minimum angle of resolution (logMAR) VA was 1.1 [Snellen 20/240, inter-quartile range (IQR) = 20/80 to 20/4000]. The median time to presentation was 7 days (IQR = 4.5 to 10). The average particulate matter 2.5 (PM2.5) concentration, a measure of air pollution, for the districts from which the patients traveled was 24.3 µg/m3 (SD = 1.6). Age- and sex-adjusted linear regression and Poisson regression results showed that higher levels of PM2.5 were associated with 0.28 worse presenting logMAR VA (Snellen 2.8 lines, P = 0.002). Patients who did not visit a VC had a 100% longer time to presentation compared to those who did (incidence rate ratio = 2.0, 95% confidence interval = 1.3-3.0, P = 0.001). Conclusion: Patient SDoH and environmental exposures can impact MK presentation. Understanding SDoH is important for public health and policy implications to mitigate eye health disparities in India.

Development of Novel Small-Molecule Activators of Pyruvate Kinase Muscle Isozyme 2, PKM2, to Reduce Photoreceptor Apoptosis.

Treatment options are lacking to prevent photoreceptor death and subsequent vision loss. Previously, we demonstrated that reprogramming metabolism via the pharmacologic activation of PKM2 is a novel photoreceptor neuroprotective strategy. However, the features of the tool compound used in those studies, ML-265, preclude its advancement as an intraocular, clinical candidate. This study sought to develop the next generation of small-molecule PKM2 activators, aimed specifically for delivery into the eye. Compounds were developed that replaced the thienopyrrolopyridazinone core of ML-265 and modified the aniline and methyl sulfoxide functional groups. Compound 2 demonstrated that structural changes to the ML-265 scaffold are tolerated from a potency and efficacy standpoint, allow for a similar binding mode to the target, and circumvent apoptosis in models of outer retinal stress. To overcome the low solubility and problematic functional groups of ML-265, compound 2's efficacious and versatile core structure for the incorporation of diverse functional groups was then utilized to develop novel PKM2 activators with improved solubility, lack of structural alerts, and retained potency. No other molecules are in the pharmaceutical pipeline for the metabolic reprogramming of photoreceptors. Thus, this study is the first to cultivate the next generation of novel, structurally diverse, small-molecule PKM2 activators for delivery into the eye.

Microbial Keratitis Isolates at a Midwestern Tertiary Eye Care Center.

PURPOSE: The aim of the study was to describe the pathogen, antimicrobial susceptibility, and trends over time of microbial keratitis (MK) at a Midwestern tertiary eye center. METHODS: Patients with MK were identified in the electronic health record from August 2012 to December 2021. Diagnostic laboratory tests with an MK diagnosis were identified and classified as laboratory positive or laboratory negative. Laboratory-positive infections were categorized as bacterial (gram-positive, gram-negative, or acid-fast bacilli), fungal, viral, Acanthamoeba , or polymicrobial. Antimicrobial susceptibilities were obtained. Trends over time were assessed using linear regression. RESULTS: Of 3288 patients with MK identified, 1012 (30.8%) had laboratory tests performed. Laboratory-positive infections (n = 499, 49.3%) were bacterial in 73.5% (n = 367) of cases, fungal in 7.8% (n = 39), viral in 1.6% (n = 8), Acanthamoeba in 1.4% (n = 7), and polymicrobial in 15.6% (n = 78). Of bacterial infections, 70% (n = 257) were gram-positive, with coagulase-negative Staphylococcus (CoNS; 31%) and Staphylococcus aureus ( S. aureus ; 23%) as the most common pathogens. Bacteria were acid-fast bacilli in 1.9% (n = 7) of cases and gram-negative in 28.1% (n = 103), with Pseudomonas aeruginosa as the predominant pathogen (47.7%). S. aureus showed antibiotic resistance from 0% (vancomycin and gentamicin) to 50% (erythromycin); CoNS from 0% (vancomycin, gentamicin, and moxifloxacin) to 64% (erythromycin). The rate of laboratory-negative MK significantly increased over time (slope estimate = 2.1% per year, P = 0.034). Rates of bacterial, fungal, viral, Acanthamoeba , and polymicrobial infections were stable over time (all slope P > 0.05). CONCLUSIONS: Bacterial keratitis accounted for most MK cases. Gram-positive bacteria were the most common isolates. CoNS and S. aureus were universally susceptible to vancomycin. Rates of MK infection types were stable over time.

Synthetic high-density lipoprotein nanoparticles delivering rapamycin for the treatment of age-related macular degeneration.

Synthetic high-density lipoprotein (sHDL) and rapamycin (Rap) have both been shown to be potential treatments for age-related macular degeneration (AMD). The low aqueous solubility of Rap, however, limits its therapeutic utility. Here we used an Apolipoprotein A-I mimetic peptide and phospholipid-based sHDL for the intravitreal delivery of Rap. By incorporation of Rap in sHDL nanoparticles (sHDL-Rap), we achieve 125-fold increase in drug aqueous concentration. When applied in vitro to retinal pigment epithelium cells, sHDL-Rap exhibited the abilities to efflux cholesterol, neutralize endotoxin, and suppress NF-κB activation. As an mTOR inhibitor, Rap induced autophagy and inhibited NF-κB-mediated pro-inflammatory signaling. Additionally, a greater reduction in lipofuscin accumulation and increased anti-inflammatory effects were achieved by sHDL-Rap relative to free drug or sHDL alone. In vivo studies demonstrated that sHDL reached the target retina pigment epithelium (RPE) layer following intravitreal administration in rats. These results suggest that sHDL-Rap holds potential as a treatment for AMD.

Safety Evaluation of Photoacoustic Tomography System for Intraocular Tumors.

Purpose: Photoacoustic tomography (PAT) has demonstrated the ability to characterize molecular components and architectural heterogeneities of intraocular tumors in enucleated human globes and in animals in vivo. Although laser safety levels have been established for illumination through the cornea, the safety limit for PAT illumination through the sclera has not been investigated. The purpose of this study is to examine if the energy level used in intraocular PAT results in ocular damage. Methods: Rabbit eyes were exposed to pulsed laser illumination at 20 mJ/cm2 at the scleral surface. Eyes were examined at 1, 7, and 28 days after the laser exposure. Examination procedures included white light and fluorescence fundus imaging, optical coherence tomography (OCT), electroretinography (ERG), and histology with hematoxylin and eosin (H&E) staining as well as terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL staining). Results: Fundus imaging and OCT of rabbit eyes at 1, 7, and 28 days following exposure of the laser illumination of the PAT system did not reveal any damage to the retinal structures. ERG showed no significant difference between the experimental and control eyes. Similarly, H&E histology did not show abnormalities in either the scleral tissue where the laser illumination was delivered or in the retinal layers. No sign of apoptosis in the layers of the retina, choroid, or optic nerve was found on TUNEL staining. Conclusions: Similar to the application of PAT to other organs, the proposed laser illumination energy level at 20 mJ/cm2 does not impose detectable harm to the ocular tissue. Translational Relevance: This study addresses illumination safety issues for PAT.

Open-Source Automatic Biomarker Measurement on Slit-Lamp Photography to Estimate Visual Acuity in Microbial Keratitis.

Purpose: To assess clinical applicability of automatic image analysis in microbial keratitis (MK) by evaluating the relationship between biomarker measurements on slit-lamp photography (SLP) and best-corrected visual acuity (BCVA). Methods: Seventy-six patients with MK with SLP images and same-day logarithm of the minimum angle of resolution (logMAR) BCVA were evaluated. MK biomarkers (stromal infiltrate, white blood cell infiltration, corneal edema, hypopyon, epithelial defect) were segmented manually by ophthalmologists and automatically by a novel, open-source, deep learning-based segmentation algorithm. Five measurements (presence, maximum width, total area, proportion of the corneal limbus area affected, centrality) were calculated. Correlations between the measurements and BCVA were calculated. An automatic regression model estimated BCVA from the measurements. Differences in performance between using manual and automatic measurements were evaluated using William's test (for correlation) and the paired-sample t-test (for absolute error). Results: Measurements had high correlations of 0.86 (manual) and 0.84 (automatic) with true BCVA. Estimated BCVA had average (mean ± SD) absolute errors of 0.39 ± 0.27 logMAR (manual, median: 0.30) and 0.35 ± 0.28 logMAR (automatic, median: 0.30) and high correlations of 0.76 (manual) and 0.80 (automatic) with true BCVA. Differences between using manual and automatic measurements were not statistically significant for correlations of measurements with true BCVA (P = .66), absolute errors of estimated BCVA (P = .15), or correlations of estimated BCVA with true BCVA (P = .60). Conclusions: The proposed algorithm measured MK biomarkers as accurately as ophthalmologists. Measurements were highly correlated with and estimative of visual acuity. Translational Relevance: This study demonstrates the potential of developing fully automatic objective and standardized strategies to aid ophthalmologists in the clinical assessment of MK.

Hexokinase 2 is dispensable for photoreceptor development but is required for survival during aging and outer retinal stress.

Photoreceptor death is the ultimate cause of vision loss in many retinal degenerative conditions. Identifying novel therapeutic avenues for prolonging photoreceptor health and function has the potential to improve vision and quality of life for patients suffering from degenerative retinal disorders. Photoreceptors are metabolically unique among other neurons in that they process the majority of their glucose via aerobic glycolysis. One of the main regulators of aerobic glycolysis is hexokinase 2 (HK2). Beyond its enzymatic function of phosphorylating glucose to glucose-6-phosphate, HK2 has additional non-enzymatic roles, including the regulation of apoptotic signaling via AKT signaling. Determining the role of HK2 in photoreceptor homeostasis may identify novel signaling pathways that can be targeted with neuroprotective agents to boost photoreceptor survival during metabolic stress. Here we show that following experimental retinal detachment, p-AKT is upregulated and HK2 translocates to mitochondria. Inhibition of AKT phosphorylation in 661W photoreceptor-like cells results in translocation of mitochondrial HK2 to the cytoplasm, increased caspase activity, and decreased cell viability. Rod-photoreceptors lacking HK2 upregulate HK1 and appear to develop normally. Interestingly, we found that HK2-deficient photoreceptors are more susceptible to acute nutrient deprivation in the experimental retinal detachment model. Additionally, HK2 appears to be important for preserving photoreceptors during aging. We show that retinal glucose metabolism is largely unchanged after HK2 deletion, suggesting that the non-enzymatic role of HK2 is important for maintaining photoreceptor health. These results suggest that HK2 expression is critical for preserving photoreceptors during acute nutrient stress and aging. More specifically, p-AKT mediated translocation of HK2 to the mitochondrial surface may be critical for protecting photoreceptors from acute and chronic stress.

Small molecule activation of metabolic enzyme pyruvate kinase muscle isozyme 2, PKM2, circumvents photoreceptor apoptosis.

Photoreceptor cell death is the ultimate cause of vision loss in many retinal disorders, and there is an unmet need for neuroprotective modalities to improve photoreceptor survival. Similar to cancer cells, photoreceptors maintain pyruvate kinase muscle isoform 2 (PKM2) expression, which is a critical regulator in aerobic glycolysis. Unlike PKM1, which has constitutively high catalytic activity, PKM2 is under complex regulation. Recently, we demonstrated that genetically reprogramming photoreceptor metabolism via PKM2-to-PKM1 substitution is a promising neuroprotective strategy. Here, we explored the neuroprotective effects of pharmacologically activating PKM2 via ML-265, a small molecule activator of PKM2, during acute outer retinal stress. We found that ML-265 increased PKM2 activity in 661 W cells and in vivo in rat eyes without affecting the expression of genes involved in glucose metabolism. ML-265 treatment did, however, alter metabolic intermediates of glucose metabolism and those necessary for biosynthesis in cultured cells. Long-term exposure to ML-265 did not result in decreased photoreceptor function or survival under baseline conditions. Notably, though, ML-265-treatment did reduce entrance into the apoptotic cascade in in vitro and in vivo models of outer retinal stress. These data suggest that reprogramming metabolism via activation of PKM2 is a novel, and promising, therapeutic strategy for photoreceptor neuroprotection.

Magnetic Hyperthermia in Y79 Retinoblastoma and ARPE-19 Retinal Epithelial Cells: Tumor Selective Apoptotic Activity of Iron Oxide Nanoparticle.

PURPOSE: To evaluate selective apoptosis of Y79 retinoblastoma versus ARPE-19 retinal pigment epithelial cells by using different doses of dextran-coated iron oxide nanoparticles (DCIONs) in a magnetic hyperthermia paradigm. METHODS: Y79 and ARPE-19 cells were exposed to different concentrations of DCIONs, namely, 0.25, 0.5, 0.75, and 1 mg/ml. After 2 hours of incubation, cells were exposed to a magnetic field with a frequency of 250 kHz and an amplitude of 4 kA/m for 30 minutes to raise the cellular temperature between 42 and 46°C. Y79 and ARPE-19 cells incubated with DCION without magnetic field exposure were used as controls. Cell viability and apoptosis were assessed at 4, 24, and 72 hours after hyperthermia treatment. RESULTS: At 4 hours following magnetic hyperthermia, cell death for Y79 cells was 1%, 8%, 17%, and 17% for 0.25, 0.5, 0.75 and 1 mg/ml of DCION, respectively. Cell death increased to 47%, 59%, 70%, and 75% at 24 hours and 16%, 45%, 50%, and 56% at 72 hours for 0.25, 0.5, 0.75, and 1 mg/ml of DCIONs, respectively. Magnetic hyperthermia did not have any significant toxic effects on ARPE-19 cells at all DCION concentrations, and minimal baseline cytotoxicity of DCIONs on Y79 and ARPE-19 cells was observed without magnetic field activation. Gene expression profiling showed that genes involved in FAS and tumor necrosis factor alpha signaling pathways were activated in Y79 cells following hyperthermia. Caspase 3/7 activity in Y79 cells increased following treatment, consistent with the activation of caspase-mediated apoptosis and loss of cell viability by magnetic hyperthermia. CONCLUSION: Magnetic hyperthermia using DCIONs selectively kills Y79 cells at 0.5 mg/ml or higher concentrations via the activation of apoptotic pathways. TRANSLATIONAL RELEVANCE: Magnetic hyperthermia using DCIONs might play a role in targeted management of retinoblastoma.

Ocular Toxicity Profile of ST-162 and ST-168 as Novel Bifunctional MEK/PI3K Inhibitors.

PURPOSE: ST-162 and ST-168 are small-molecule bifunctional inhibitors of MEK and PI3K signaling pathways that are being developed as novel antitumor agents. Previous small-molecule and biologic MEK inhibitors demonstrated ocular toxicity events that were dose limiting in clinical studies. We evaluated in vitro and in vivo ocular toxicity profiles of ST-162 and ST-168. METHODS: Photoreceptor cell line 661W and adult retinal pigment epithelium cell line ARPE-19 were treated with increasing concentrations of bifunctional inhibitors. Western blots, cell viability, and caspase activity assays were performed to evaluate MEK and PI3K inhibition and dose-dependent in vitro toxicity, and compared with monotherapy. In vivo toxicity profile was assessed by intravitreal injection of ST-162 and ST-168 in Dutch-Belted rabbits, followed by ocular examination and histological analysis of enucleated eyes. RESULTS: Retinal cell lines treated with ST-162 or ST-168 exhibited dose-dependent inhibition of MEK and PI3K signaling. Compared with inhibition by monotherapies and their combinations, bifunctional inhibitors demonstrated reduced cell death and caspase activity. In vivo, both bifunctional inhibitors exhibited a more favorable toxicity profile when compared with MEK inhibitor PD0325901. CONCLUSIONS: Novel MEK and PI3K bifunctional inhibitors ST-162 and ST-168 demonstrate favorable in vitro and in vivo ocular toxicity profiles, supporting their further development as potential therapeutic agents targeting multiple aggressive tumors.

Photoreceptor metabolic reprogramming provides survival advantage in acute stress while causing chronic degeneration.

Photoreceptor death is the root cause of vision loss in many retinal disorders, and there is an unmet need for neuroprotective modalities to improve photoreceptor survival. The biosynthetic requirement of photoreceptors is among the highest in the body, and to meet this demand, photoreceptors maintain their ability to perform aerobic glycolysis. This highly regulated form of glycolysis allows cells to efficiently budget their metabolic needs and may be a critical link between photoreceptor function and survival. Pyruvate kinase muscle isozyme 2 (PKM2) is a key regulator of aerobic glycolysis. In the present study, we characterized the effect of PKM2 deletion on baseline functioning and survival of photoreceptors over time by utilizing a photoreceptor-specific, PKM2 knockout mouse model. We found that upon PKM2 deletion, PKM1 is upregulated in the outer retina and there is increased expression of genes involved in glucose metabolism, which led to chronic degenerative changes in the outer retina of these mice. We also discovered that this metabolic reprogramming provided a survival advantage to photoreceptors in an experimental model of retinal detachment. This study strongly supports the hypothesis that reprogramming metabolism may be a novel therapeutic strategy for photoreceptor neuroprotection during acute stress.

FAS apoptotic inhibitory molecule 2 is a stress-induced intrinsic neuroprotective factor in the retina.

We report the neuroprotective role of FAS apoptotic inhibitory molecule 2 (FAIM2), an inhibitor of the FAS signaling pathway, during stress-induced photoreceptor apoptosis. Retinal detachment resulted in increased FAIM2 levels in photoreceptors with higher amounts detected at the tips of outer segments. Activation of FAS death receptor via FAS-ligand led to JNK-mediated FAIM2 phosphorylation, decreased proteasome-mediated degradation and increased association with the FAS receptor. Photoreceptor apoptosis was accelerated in Faim2 knockout mice following experimental retinal detachment. We show that FAIM2 is primarily involved in reducing stress-induced photoreceptor cell death but this effect was transient. FAIM2 was found to interact with both p53 and HSP90 following the activation of the FAS death pathway and FAIM2/HSP90 interaction was dependent on the phosphorylation of FAIM2. Lack of FAIM2 led to increased expression of proadeath genes Fas and Ripk1 in the retina under physiologic conditions. These results demonstrate that FAIM2 is an intrinsic neuroprotective factor activated by stress in photoreceptors and delays FAS-mediated photoreceptor apoptosis. Modulation of this pathway to increase FAIM2 expression may be a potential therapeutic option to prevent photoreceptor death.

Translocation and association of ROCK-II with RhoA and HSP27 during contraction of rabbit colon smooth muscle cells.

The focus of the paper is to understand the role of HSP27 in mediating the association of RhoA with ROCK-II in sustained contraction of smooth muscle cells from the rabbit colon. In circular smooth muscle cells; acetylcholine-induced contraction (10(-7)M) was associated with translocation of ROCK-II to the particulate fraction, which remained sustained at 4 min after stimulation (135.1+/-8.1% increase, P

Direct association and translocation of PKC-alpha with calponin.

Calponin has been implicated in the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated MgATPase activity of phosphorylated myosin. Calponin has also been shown to interact with PKC. We have studied the interaction of calponin with PKC-alpha and with the low molecular weight heat-shock protein (HSP)27 in contraction of colonic smooth muscle cells. Particulate fractions from isolated smooth muscle cells were immunoprecipitated with antibodies to calponin and Western blot analyzed with antibodies to HSP27 and to PKC-alpha. Acetylcholine induced a sustained increase in the immunocomplexing of calponin with HSP27 and of calponin with PKC-alpha in the particulate fraction, indicating an association of the translocated proteins in the membrane. To examine whether the observed interaction in vivo is due to a direct interaction of calponin with PKC-alpha, a cDNA of 1.3 kb of human calponin gene was PCR amplified. PCR product encoding 622 nt of calponin cDNA (nt 351-972 corresponding to amino acids 92-229) was expressed as fusion glutathione S-transferase (GST) protein in the vector pGEX-KT. We have studied the direct association of GST-calponin fusion protein with recombinant PKC-alpha in vitro. Western blot analysis of the fractions collected after elution with reduced glutathione buffer (pH 8.0) show a coelution of GST-calponin with PKC-alpha, indicating a direct association of GST-calponin with PKC-alpha. These data suggest that there is a direct association of translocated calponin and PKC-alpha in the membrane and a role for the complex calponin-PKC-alpha-HSP27, in contraction of colonic smooth muscle cells.

Phosphorylated HSP27 essential for acetylcholine-induced association of RhoA with PKCalpha.

Reorganization of the cytoskeleton and association of contractile proteins are important steps in modulating smooth muscle contraction. Heat shock protein (HSP) 27 has significant effects on actin cytoskeletal reorganization during smooth muscle contraction. We investigated the role of phosphorylated HSP27 in modulating acetylcholine-induced sustained contraction of smooth muscle cells from the rabbit colon by transfecting smooth muscle cells with phosphomimic (3D) or nonphosphomimic (3G) HSP27. In 3G cells, the initial peak contractile response at 30 s was inhibited by 25% (24.0 +/- 4.5% decrease in cell length, n = 4). The sustained contraction was greatly inhibited by 75% [9.3 +/-.9% decreases in cell length (n = 4)]. Furthermore, in 3D cells, translocation of both PKCalpha and of RhoA was greatly enhanced and resulted in a greater association of PKCalpha-RhoA in the membrane fraction. In 3G transfected cells, PKCalpha and RhoA failed to translocate in response to stimulation with acetylcholine, resulting in an inhibition of association of PKCalpha-RhoA in the membrane fraction. Studies using GST-RhoA fusion protein indicate that there is a direct association of RhoA with PKCalpha and with HSP27. The results suggest that phosphorylated HSP27 plays a crucial role in the maintenance of association of PKCalpha-RhoA in the membrane fraction and in the maintenance of acetylcholine-induced sustained contraction.