Relationship of Nocturnal Insomnia Symptoms and Outcomes After Hypoglossal Nerve Stimulation.

Objective: In patients undergoing hypoglossal nerve stimulation (HGNS), we examined the Insomnia Severity Index (ISI) to understand how baseline sleep onset insomnia (SOI), sleep maintenance insomnia (SMI), and early morning awakening (EMA) affected postsurgical outcomes. Study Design: Observational. Setting: Multicenter registry. Methods: We included patients from the Adherence and Outcomes of Upper Airway Stimulation for Obstructive Sleep Apnea International Registry (ADHERE) with a baseline ISI from 2020 to 2023. Regression analysis examined the association of ISI question scores for SOI, SMI, and EMA and outcomes: Apnea-Hypopnea Index (AHI) reduction, device usage, changes in the Epworth Sleepiness Scale (ESS) and overall ISI score, final visit (FV) completion, and satisfaction. Results: No relationship was noted between insomnia subtypes and AHI reduction or FV completion. In the subgroup of patients with baseline moderate/severe insomnia, patients with major impairment for SOI used their device 64 min/day longer than those with minimal impairment. Among all patients, those with baseline major impairment for SOI had a 2.3 points greater improvement in ISI from baseline to FV compared to patients with minimal impairment, while patients with baseline major impairment for SMI had a 2.0 and 3.5 points greater improvement in the ESS and ISI than those with minimal impairment. Patients with EMA and moderate/severe baseline insomnia had decreased odds of being satisfied after surgery. Conclusion: In ADHERE, nocturnal symptoms of insomnia did not limit HGNS efficacy or therapy use. Conversely, those with worse insomnia subtype impairments at baseline had improved outcomes related to adherence, sleepiness, and insomnia at the FV.

Impact of Insomnia on Hypoglossal Nerve Stimulation Outcomes in the ADHERE Registry.

OBJECTIVE: We aimed to determine the preoperative prevalence of insomnia in the Adherence and Outcomes of Upper Airway Stimulation for OSA International Registry (ADHERE) and to examine serial sleep-related data longitudinally, in particular the Insomnia Severity Index (ISI), to compare outcomes between patients with no/subthreshold insomnia (ISI < 15) and moderate/severe insomnia (ISI ≥ 15) at baseline. METHODS: We analyzed observational data from ADHERE between March 2020 and September 2022. Baseline demographic and mental health (MH) data, apnea hypopnea index (AHI), ISI, and ESS (Epworth Sleepiness Scale) were recorded. At post-titration (PT) and final visits, AHI, ISI, ESS and nightly usage were compared between baseline ISI < 15 and ISI ≥ 15 subgroups. RESULTS: A baseline ISI was obtained in 928 patients (62% with ISI ≥ 15). Of the 578 and 141 patients reaching the 12- and 24-month time periods to complete PT and final visits, 292 (50.5%) and 91 (64.5%) completed the ISI, respectively. Baseline MH conditions were higher with ISI ≥ 15 than ISI < 15 (p < 0.001). AHI reduction and adherence did not differ between patients with baseline ISI ≥ 15 and ISI < 15. Patients with ISI ≥ 15 experienced greater improvement in ESS than ISI < 15 at post-titration and final visits (p = 0.014, 0.025). All patients had improved nocturnal, daytime, and overall ISI scores at follow-up visits (p < 0.001), especially for those with baseline ISI ≥ 15 compared with ISI < 15 (p < 0.05). CONCLUSION: HGNS therapy efficacy and adherence were similar between ISI severity subgroups at follow-up visits. Insomnia and sleepiness scores improved in all patients with HGNS therapy and to a greater degree in patients with baseline moderate/severe insomnia. LEVEL OF EVIDENCE: 4 Laryngoscope, 134:471-479, 2024.

A Noninferiority Analysis of 3- vs 2-Incision Techniques for Hypoglossal Nerve Stimulator Implantation.

OBJECTIVE: The only hypoglossal nerve stimulation (HNS) device available for US clinical use is implanted through 3 incisions. A recently proposed 2-incision modification moved the respiratory sensing lead from the fifth to the second intercostal space to eliminate the third lower chest incision. This study compared perioperative data and therapeutic outcomes between the techniques. STUDY DESIGN: Noninferiority cohort analysis of a retrospective and prospective registry study. SETTING: Tertiary care and community surgical centers. METHODS: Patients with obstructive sleep apnea underwent HNS implantation via a modified 2-incision technique (I2). A cohort previously implanted via the standard 3-incision technique (I3) were 1:1 propensity score matched for a noninferiority analysis of postoperative outcomes. RESULTS: There were 404 I3 patients and 223 I2 patients across 6 participating centers. Operative time decreased from 128.7 minutes (95% CI, 124.5-132.9) in I3 patients to 86.6 minutes (95% CI, 83.7-97.6) in I2 patients (P < .001). Postoperative sleep study data were available for 76 I2 patients who were matched to I3 patients. The change in apnea-hypopnea index between the cohorts was statistically noninferior (a priori noninferiority margin: 7.5 events/h; mean difference, 1.51 [97.5% CI upper bound, 5.86]). There were no significant differences between the cohorts for baseline characteristics, perioperative adverse event rates, or change in Epworth Sleepiness Score (P > .05). CONCLUSION: In a multicenter registry, a 2-incision implant technique for a commercially available HNS device had a statistically noninferior therapeutic efficacy profile when compared with the standard 3-incision approach. The 2-incision technique is safe and effective for HNS implantation.

Underrepresented Women Leaders: Lasting Impact of Gender Homophily in Surgical Faculty Networks.

OBJECTIVE: Despite the increase of women in surgical fields and resources toward advancing women, there is no corresponding increased representation of women in higher academic ranks and leadership. The following hypotheses are explored: 1) men and women build/maintain different relationship networks and 2) women are not similarly included within the organization and do not receive the same sponsorship as male counterparts. STUDY DESIGN: Prospective observational study. METHODS: Three months of prospective, passive organizational network data of full-time faculty at an academic medical center were collected by analysis of deidentified internal email communication logs. Data were analyzed and strength of network relationships was assessed using algorithms measuring the tie, or connection, score. Data analysis was performed with standard statistical methods and multivariable regression models, comparing network relationships based on gender and academic rank. RESULTS: Among 345 full-time faculties from surgical departments, 45.2% were female Assistant Professors, but only 9.8% were female full Professors. Men had 55% more network relationships with other men than women had with men. Gender homophily was particularly pronounced at the higher academic ranks. Men compared to women in higher ranks had 157% more network relationships to other men in lower ranks. Multivariable regression models suggested direct association of these gender differences in relationships with more women in lower academic ranks. CONCLUSION: Higher academic rank can be predicted by male gender, tenure, and number of meaningful relationships. Women are underrepresented at the leadership level in surgical departments. Gender homophily is present in collaboration networks among academic surgeons and is associated with impeded female career advancement. LEVEL OF EVIDENCE: 2 Laryngoscope, 132:20-25, 2022.

Impact of Body Mass Index and Discomfort on Upper Airway Stimulation: ADHERE Registry 2020 Update.

OBJECTIVES/HYPOTHESIS: To provide the ADHERE registry Upper Airway Stimulation (UAS) outcomes update, including analyses grouped by body mass index (BMI) and therapy discomfort. STUDY DESIGN: Prospective observational study. METHODS: ADHERE captures UAS outcomes including apnea-hypopnea index (AHI), Epworth sleepiness scale (ESS), therapy usage, patient satisfaction, clinician assessment, and safety over a 1-year period. BMI ≤32 kg/m2 (BMI32 ) and 32 < BMI ≤35 kg/m2 (BMI35 ) group outcomes were examined. RESULTS: One thousand eight hundred forty-nine patients enrolled in ADHERE, 1,019 reached final visit, 843 completed the visit. Significant changes in AHI (-20.9, P < .0001) and ESS (- 4.4, P < .0001) were demonstrated. Mean therapy usage was 5.6 ± 2.2 hr/day. Significant therapy use difference was present in patients with reported discomfort versus no discomfort (4.9 ± 2.5 vs. 5.7 ± 2.1 hr/day, P = .01). Patients with discomfort had higher final visit mean AHI versus without discomfort (18.9 ± 18.5 vs. 13.5 ± 13.7 events/hr, P = .01). Changes in AHI and ESS were not significantly different. Serious adverse events reported in 2.3% of patients. Device revision rate was 1.9%. Surgical success was less likely in BMI35 versus BMI32 patients (59.8% vs. 72.2%, P = .02). There was a significant therapy use difference: 5.8 ± 2.0 hr/day in BMI32 versus 5.2 ± 2.2 hr/day in BMI35 (P = .028). CONCLUSIONS: Data from ADHERE demonstrate high efficacy rates for UAS. Although surgical response rate differs between BMI32 and BMI35 patient groups, the AHI and ESS reduction is similar. Discomfort affects therapy adherence and efficacy. Thus, proper therapy settings adjustment to ensure comfort is imperative to improve outcomes. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:2616-2624, 2021.

Improving outcomes of hypoglossal nerve stimulation therapy: current practice, future directions, and research gaps. Proceedings of the 2019 International Sleep Surgery Society Research Forum.

Hypoglossal nerve stimulation (HGNS) has evolved as a novel and effective therapy for patients with moderate-to-severe obstructive sleep apnea. Despite positive published outcomes of HGNS, there exist uncertainties regarding proper patient selection, surgical technique, and the reporting of outcomes and individual factors that impact therapy effectiveness. According to current guidelines, this therapy is indicated for select patients, and recommendations are based on the Stimulation Therapy for Apnea Reduction or STAR trial. Ongoing research and physician experiences continuously improve methods to optimize the therapy. An understanding of the way in which airway anatomy, obstructive sleep apnea phenotypes, individual health status, psychological conditions, and comorbid sleep disorders influence the effectiveness of HGNS is essential to improve outcomes and expand therapy indications. This article presents discussions on current evidence, future directions, and research gaps for HGNS therapy from the 10th International Surgical Sleep Society expert research panel. CITATION: Suurna MV, Jacobowitz O, Chang J, et al. Improving outcomes of hypoglossal nerve stimulation therapy: current practice, future directions and research gaps. Proceedings of the 2019 International Sleep Surgery Society Research Forum. J Clin Sleep Med. 2021;17(12):2477-2487.

Obstructive Sleep Apnea: Non-positive Airway Pressure Treatments.

Undiagnosed and untreated obstructive sleep apnea (OSA) is associated with health comorbidities and negatively affects quality of life. Alternative treatments should be considered in patients who are unable to tolerate or benefit from positive airway pressure treatment. When properly indicated, positional devices, oral appliances, airway surgery, and hypoglossal nerve stimulation have been shown to be effective in treating OSA. Hypoglossal nerve stimulation is a successful second-line treatment with low associated morbidity and complication rate.

Drug-Induced Sleep Endoscopy and Hypoglossal Nerve Stimulation Outcomes: A Multicenter Cohort Study.

OBJECTIVES/HYPOTHESIS: To determine the association between findings of blinded reviews of preoperative drug-induced sleep endoscopy (DISE) and outcomes of hypoglossal nerve stimulation (HNS) for obstructive sleep apnea (OSA). STUDY DESIGN: Cohort study. METHODS: A retrospective, multicenter cohort study of 343 adults who underwent treatment of OSA with HNS from 10 academic medical centers was performed. Preoperative DISE videos were scored by four blinded reviewers using the VOTE Classification and evaluation of a possible primary structure contributing to airway obstruction. Consensus DISE findings were examined for an association with surgical outcomes based on therapy titration polysomnogram (tPSG). Treatment response was defined by a decrease of ≥50% in the apnea-hypopnea index (AHI) to <15 events/hour. RESULTS: Study participants (76% male, 60.4 ± 11.0 years old) had a body mass index of 29.2 ± 3.6 kg/m2 . AHI decreased (35.6 ± 15.2 to 11.0 ± 14.1 events/hour; P < .001) on the tPSG, with a 72.6% response rate. Complete palate obstruction (vs. none) was associated with the greatest difference in AHI improvement (-26.8 ± 14.9 vs. -19.2 ± 12.8, P = .02). Complete (vs. partial/none) tongue-related obstruction was associated with increased odds of treatment response (78% vs. 68%, P = .043). Complete (vs. partial/none) oropharyngeal lateral wall-related obstruction was associated with lower odds of surgical response (58% vs. 74%, P = .042). CONCLUSIONS: The DISE finding of primary tongue contribution to airway obstruction was associated with better outcomes, whereas the opposite was true for the oropharyngeal lateral walls. This study suggests that the role for DISE in counseling candidates for HNS extends beyond solely for excluding complete concentric collapse related to the velum. LEVEL OF EVIDENCE: 3 Laryngoscope, 131:1676-1682, 2021.

Establishing an Office-Based Framework for Resuming Otolaryngology Care in Academic Practice During the COVID-19 Pandemic.

OBJECTIVE: The COVID-19 health crisis abruptly disrupted the practice of otolaryngology. This article aims to define the changes needed to operate an academic otolaryngology practice safely and efficiently from within the epicenter of the pandemic. We define the areas of normal patient workflow that have been affected by COVID-19, and we offer mitigation strategies with attention paid to the specific needs of subspecialties. DATA SOURCES: The article includes data specific to the office practice metrics of the Weill Cornell Medicine Department of Otolaryngology-Head and Neck Surgery, as well as publically available data from New York Presbyterian Hospital system and the New York Times. REVIEW METHODS: Expert opinion. CONCLUSIONS: Through careful planning and execution, it is possible to reestablish safe otolaryngologic patient care during the COVID-19 pandemic. It will require a significant change from prior practice models for successful implementation. Additionally, telemedicine can be positively integrated into the treatment of otolaryngology diseases for new and established patients. IMPLICATIONS FOR PRACTICE: The information conveyed in this review can be used as a guide by large and small otolaryngology groups to identify aspects of the patient visit that are "at risk" due to COVID-19, and it suggests sensible responses that can be made without a significant disruption to normal practice. The methods used to identify vulnerabilities with the patient visit process can be applied to future unforeseen crises, such as a resurgence of COVID-19 or a novel pandemic.

Intraoperative identification of mixed activation profiles during hypoglossal nerve stimulation.

STUDY OBJECTIVES: The effectiveness of hypoglossal nerve stimulation (HGNS) in the treatment of obstructive sleep apnea (OSA) depends on the selective stimulation of nerve fibers that innervate the tongue muscles that produce tongue protrusion (genioglossus) and stiffening (transverse/vertical) while avoiding fibers that innervate muscles that produce tongue retraction (styloglossus/hyoglossus). Postoperative treatment failures can be related to mixed activation of retractor and protrusor muscles, despite intraoperative efforts to identify and avoid nerve fibers that innervate the retractor muscles. This study describes a novel intraoperative protocol that more optimally identifies mixed activation by utilizing an expanded set of stimulation/recording parameters. METHODS: This study was a case series in a university hospital setting of patients undergoing unilateral hypoglossal nerve stimulation implantation for obstructive sleep apnea. Data included electromyographic responses in the genioglossus and styloglossus/hyoglossus to intraoperative stimulation with an implantable pulse generator using unipolar (- - -, o-o) and bipolar (+-+) settings. RESULTS: In a subset of patients (3/55), low-intensity unipolar implantable pulse generator stimulation revealed significant mixed activation of the styloglossus/hyoglossus and genioglossus muscles that was not evident under standard bipolar implantable pulse generator stimulation conditions. Additional surgical dissection and repositioning of the electrode stimulation cuff reduced mixed activation. CONCLUSIONS: A novel intraoperative neurophysiological monitoring protocol was able to detect significant mixed activation during hypoglossal nerve stimulation that was otherwise absent using standard parameters. This enabled successful electrode cuff repositioning and a dramatic reduction of mixed activation.

Contralateral Tongue Muscle Activation during Hypoglossal Nerve Stimulation.

OBJECTIVE: The effectiveness of upper airway stimulation via hypoglossal nerve stimulation for obstructive sleep apnea depends upon the pattern of tongue muscle activation produced. This study investigated the nature of contralateral tongue muscle activation by unilateral hypoglossal nerve stimulation using intraoperative nerve integrity monitoring in conjunction with electromyography and explored the relationship between contralateral tongue muscle activation and polysomnographic measures of obstructive sleep apnea severity. STUDY DESIGN: Prospective case series. SETTING: Tertiary care medical center. SUBJECTS AND METHODS: Fifty-one patients underwent unilateral (right) hypoglossal nerve stimulator implantation for obstructive sleep apnea. Neurophysiological data included electromyographic responses in ipsilateral (right) and contralateral (left) genioglossus muscles in response to intraoperative bipolar probe stimulation (0.3 mA) of medial hypoglossal nerve branches. Clinical data included pre- and postoperative apnea-hypopnea indices and oxygen desaturation levels. RESULTS: A subset of patients (20/51, 39%) exhibited electromyographic responses in both the ipsilateral and contralateral genioglossus (bilateral), whereas the remaining patients (31/51, 61%) exhibited electromyographic responses only in the ipsilateral genioglossus (unilateral). The baseline characteristics of bilateral and unilateral responders were similar. Both groups exhibited significant and comparable improvements in apnea-hypopnea index and oxygen desaturations after hypoglossal nerve stimulation. Neither the amplitude nor the latency of contralateral genioglossus responses was predictive of clinical outcomes. CONCLUSION: A subset of patients undergoing unilateral hypoglossal nerve stimulation exhibits activation of contralateral genioglossus muscles. Patients with unilateral and bilateral genioglossus responses exhibit comparable, robust improvements in apnea-hypopnea index and oxygen desaturation levels.

Upper-Airway Stimulation Before, After, or Without Uvulopalatopharyngoplasty: A Two-Year Perspective.

OBJECTIVE: Upper airway stimulation (UAS) is an effective second-line treatment for obstructive sleep apnea (OSA). In certain patients, there is a considerable need for advanced programming, notably with inadequate palatal response to therapy. The aim of the study was to investigate the impact of uvulopalatopharyngoplasty and tonsillectomy (UPPP-TE) on UAS therapy outcomes from a 2-year perspective after implantation. METHODS: This study included all consecutive patients implanted with UAS in which a full set of 1- and 2-year follow-up assessments (M12 and M24) were obtained. Cases were analyzed in three groups: patients with UPPP-TE after (group 1) and before (group 2) UAS, and those without UPPP-TE (group 3). RESULTS: Therapy success could be achieved in about 80% of the entire cohort. Groups 2 and 3 did not differ significantly with regard to obesity, Apnea-Hypopnea Index, or Oxygen Desaturation Index. With regard to initial sleep endoscopy, there were fewer patients without any obstruction at the palatal and oropharyngeal levels and higher prevalence of lateral obstruction patterns at oropharynx in group 1 in contrast to groups 2 and 3. Groups 2 and 3 showed similar results, although group 2 patients underwent UPPP-TE before UAS implantation. CONCLUSION: UPPP-TE should be considered in patients with persistent OSA after UAS implantation if the obstruction is identified at the level of velum and oropharynx. Although this approach has higher response rates and better outcomes can be achieved in patients with UAS, there is no indication for patients to routinely undergo UPPP-TE prior to UAS implantation. LEVEL OF EVIDENCE: 4 Laryngoscope, 129:514-518, 2019.

Hypoglossal nerve stimulation in three adults with down syndrome and severe obstructive sleep apnea.

We present three adults with Down syndrome and obstructive sleep apnea (OSA) treated with hypoglossal nerve stimulation. The average age of these three males was 37.3 years. All patients had severe OSA on polysomnography. Postimplantation titration sleep studies exhibited residual OSA according to average total apnea-hypopnea index (AHI), but there were significant reductions (86%-100%) in the titrated AHI at the ideal device setting. Morbidity of the surgery was low, and there was excellent device adherence, with an average of 57.3 hours/week. All patients reported subjective improvements in symptoms, and one patient experienced improved blood sugar control. Laryngoscope, 129:E402-E406, 2019.

A case of hypoglossal nerve stimulator-resistant obstructive sleep apnea cured with the addition of a chin strap.

A population of appropriately selected patients does not respond, or does not achieve cure, with hypoglossal nerve stimulation (HGNS). We describe the case of nonresponder whose obstructive sleep apnea (OSA) resolved with the addition of chin strap. After initial placement and titration of HGNS implant, follow-up sleep study demonstrated persistent moderate OSA. Drug-induced sleep endoscopy demonstrated supraglottic collapse with activate neurostimulation. With mouth closure and change of stimulation settings to unipolar from bipolar, the airway collapse and desaturations improved. The follow-up polysomnogram with (HGNS) therapy and chin strap demonstrated resolution of sleep apnea. Laryngoscope, 128:1727-1729, 2018.

Mucosal contact points and paranasal sinus pneumatization: Does radiology predict headache causality?

OBJECTIVES/HYPOTHESIS: The goal of this study was to evaluate the prevalence of mucosal contact points (MCP), concha bullosa (CB), and variable paranasal sinus (PNS) volumes among patients sent for rhinogenic headache workup as compared with controls. STUDY DESIGN: Retrospective study with case and control groups. METHODS: Fifty-three adults with clear PNS computed tomography (CT) scans were included: 28 patients who have originally undergone PNS CT scan as part of sinus (rhinogenic) headache workup, and 25 controls in whom PNS CT scans were obtained for other purposes. All subjects were asked to report their symptoms using a headache scoring system. CT scans of all subjects were analyzed for presence of MCP, CB, as well as for volumes of maxillary, frontal, and sphenoid sinuses. RESULTS: MCP was found in 40% of controls, and in 50%, 50%, and 40% of mild, moderate, and severe headache groups, respectively. CB was found in 24% of controls, and in 33.3%, 58.3%, and 20%, of mild, moderate, and severe headache groups, respectively. Total volume of the measured PNS ranged from 23.9 to 81.4 cm(3) (mean ± standard deviation [SD], 48.3 ± 15.8) in the control group and from 5.31 to 87.4 cm(3) (mean ± SD, 43.6 ± 16) in the patient group. No statistically significant difference was found between groups regarding all studied variables. CONCLUSIONS: Radiological identification of MCP, CB, or hyperpneumatized sinuses does not seem to be a predictor of headache causality. Further studies are required to identify clinical scenarios in which these variations may contribute to pain symptoms. LEVEL OF EVIDENCE: 3b.

Randomized placebo-controlled trial of pantoprazole for daytime sleepiness in GERD and obstructive sleep disordered breathing.

OBJECTIVE: To determine the efficacy of pantoprazole therapy for daytime somnolence, psychomotor vigilance, and quality of life in patients with mild-moderate obstructive sleep disordered breathing (OSDB) and gastroesophageal reflux disease (GERD). STUDY DESIGN: Randomized, double-blind, placebo-controlled crossover trial. METHODS: Sixty patients with daytime sleepiness, mild-moderate OSDB and GERD were randomly assigned a 2-week treatment with pantoprazole 40 mg or placebo followed by a 2-week washout period and crossover respectively to 2-week treatment with placebo or pantoprazole. Outcomes included Epworth Sleepiness Score (ESS), sleep-related quality-of-life (FOSQ), and reaction time. RESULTS: With pantoprazole, patients reported statistically significantly greater improvement of overall reflux symptoms (P = 0.0003) and in ESS (P = 0.04). A significant improvement was noted in FOSQ for both treatments with a trend toward greater improvement with pantoprazole (P = 0.058). No improvement in reaction times was observed. CONCLUSION: Patients with coexistent GERD and OSDB noted significant improvement in daytime sleepiness after treatment with pantoprazole over placebo likely related to a reduction in nocturnal reflux-related arousals.

Cofilin mediates ATP depletion-induced endothelial cell actin alterations.

Ischemia and sepsis lead to endothelial cell damage, resulting in compromised microvascular flow in many organs. Much remains to be determined regarding the intracellular structural events that lead to endothelial cell dysfunction. To investigate potential actin cytoskeletal-related mechanisms, ATP depletion was induced in mouse pancreatic microvascular endothelial cells (MS1). Fluorescent imaging and biochemical studies demonstrated a rapid and progressive increase in F-actin along with a decrease in G-actin at 60 min. Confocal microscopic analysis showed ATP depletion resulted in destruction of actin stress fibers and accumulation of F-actin aggregates. We hypothesized these actin alterations were secondary to dephosphorylation/activation of actin-depolymerizing factor (ADF)/cofilin proteins. Cofilin, the predominant isoform expressed in MS1 cells, was rapidly dephosphorylated/activated during ATP depletion. To directly investigate the role of cofilin activation on the actin cytoskeleton during ischemia, MS1 cells were infected with adenoviruses containing the cDNAs for wild-type Xenopus laevis ADF/cofilin green fluorescent protein [XAC(wt)-GFP], GFP, and the constitutively active and inactive isoforms XAC(S3A)-GFP and XAC(S3E)-GFP. The rate and extent of cortical actin destruction and actin aggregate formation were increased in ATP-depleted XAC(wt)-GFP- and XAC(S3A)-GFP-expressing cells, whereas increased actin stress fibers were observed in XAC(S3E)-GFP-expressing cells. To investigate the upstream signaling pathway of ADF/cofilin, LIM kinase 1-GFP (LIMK1-GFP) was expressed in MS1 cells. Cells expressing LIMK1-GFP protein had higher levels of phosphorylated ADF/cofilin, increased stress fibers, and delayed F-actin cytoskeleton destruction during ATP depletion. These results strongly support the importance of cofilin regulation in ischemia-induced endothelial cell actin cytoskeleton alterations leading to cell damage and microvascular dysfunction.