Defining the Cerebral Cavernous Malformation Protein Signaling Network in Endothelial Cells

Cerebral cavernous malformations (CCM) are cerebrovascular lesions occurring with homozygous loss of function mutations of CCM1, -2, and -3. CCMs can cause neurological deficits, seizures, and fatal hemorrhagic stroke. Loss of function in any one of the three CCM proteins leads to defects in in vitro angiogenesis assays, along with increased actin stress fibers in endothelial cells. Elevated RhoA and Rho kinase (ROCK)-LIM kinase hyperactivity results upon loss of the CCM proteins, likely from dysregulation of the E3 ubiquitin ligase SMURF1. Phosphorylation of the LIM kinase substrate and actin depolymerizing factor cofilin is increased in primary endothelial cells and surgically resected human CCM lesions, providing a novel biomarker for CCM disease. The CCM phenotype and RhoA-ROCK hyperactivity can be recapitulated in endothelial progenitor-derived endothelial cells, primary cells that can be isolated from the peripheral blood of human CCM patients. Significantly, treatment with structurally distinct small molecule ROCK inhibitors rescues the CCM phenotype in vitro and reverses the hyperactivation of ROCK effectors seen with loss of the CCM proteins. Interrogation of the kinome in endothelial cells with CCM2 knockdown using multiplexed kinase inhibitor beads coupled to mass spectroscopy reveals striking dysregulation, likely multifactorial from loss of the scaffold-like CCM2 protein, dysregulation of SMURF1, and concomitant hyperactivation of the small GTPase RhoA. Of particular interest are the loss of two critical angiogenesis kinases, TIE-2 and BMX. Together, these data provides insight to the molecular mechanism of CCM pathophysiology and provides clues for future successful pharmacological treatment of the disease.Doctor of Philosoph

Derivation and validation of a clinical prediction rule for upper limb functional outcomes after traumatic cervical spinal cord injury

IMPORTANCE: Traumatic cervical spinal cord injury (SCI) can result in debilitating paralysis. Following cervical SCI, accurate early prediction of upper limb recovery can serve an important role in guiding the appropriateness and timing of reconstructive therapies. OBJECTIVE: To develop a clinical prediction rule to prognosticate upper limb functional recovery after cervical SCI. DESIGN, SETTING, AND PARTICIPANTS: This prognostic study was a retrospective review of a longitudinal cohort study including patients enrolled in the National SCI model systems (SCIMS) database in US. Eligible patients were 15 years or older with tetraplegia (neurological level of injury C1-C8, American Spinal Cord Injury Association [ASIA] impairment scale [AIS] A-D), with early (within 1 month of SCI) and late (1-year follow-up) clinical examinations from 2011 to 2016. The data analysis was conducted from September 2021 to June 2022. MAIN OUTCOMES AND MEASURES: The primary outcome was a composite of dependency in eating, bladder management, transfers, and locomotion domains of functional independence measure at 1-year follow-up. Each domain ranges from 1 to 7 with a lower score indicating greater functional dependence. Composite dependency was defined as a score of 4 or higher in at least 3 chosen domains. Multivariable logistic regression was used to predict the outcome based on early neurological variables. Discrimination was quantified using C statistics, and model performance was internally validated with bootstrapping and 10-fold cross-validation. The performance of the prediction score was compared with AIS grading. Data were split into derivation (2011-2014) and temporal-validation (2015-2016) cohorts. RESULTS: Among 2373 patients with traumatic cervical SCI, 940 had complete 1-year outcome data (237 patients [25%] aged 60 years or older; 753 men [80%]). The primary outcome was present in 118 patients (13%), which included 92 men (78%), 83 (70%) patients who were younger than 60 years, and 73 (62%) patients experiencing AIS grade A SCI. The variables significantly associated with the outcome were age (age 60 years or older: OR, 2.31; 95% CI, 1.26-4.19), sex (men: OR, 0.60; 95% CI, 0.31-1.17), light-touch sensation at C5 (OR, 0.44; 95% CI, 0.44-1.01) and C8 (OR, 036; 95% CI, 0.24-0.53) dermatomes, and motor scores of the elbow flexors (C5) (OR, 0.74; 95% CI, 0.60-0.89) and wrist extensors (C6) (OR, 0.61; 95% CI, 0.49-0.75). A multivariable model including these variables had excellent discrimination in distinguishing dependent from independent patients in the temporal-validation cohort (C statistic, 0.90; 95% CI, 0.88-0.93). A clinical prediction score (range, 0 to 45 points) was developed based on these measures, with higher scores increasing the probability of dependency. The discrimination of the prediction score was significantly higher than from AIS grading (change in AUC, 0.14; 95% CI, 0.10-0.18; P \u3c .001). CONCLUSIONS AND RELEVANCE: The findings of this study suggest that this prediction rule may help prognosticate upper limb function following cervical SCI. This tool can be used to set patient expectations, rehabilitation goals, and aid decision-making regarding the appropriateness and timing for upper limb reconstructive surgeries

Upper limb nerve transfer surgery in patients with tetraplegia

IMPORTANCE: Cervical spinal cord injury (SCI) causes devastating loss of upper extremity function and independence. Nerve transfers are a promising approach to reanimate upper limbs; however, there remains a paucity of high-quality evidence supporting a clinical benefit for patients with tetraplegia. OBJECTIVE: To evaluate the clinical utility of nerve transfers for reanimation of upper limb function in tetraplegia. DESIGN, SETTING, AND PARTICIPANTS: In this prospective case series, adults with cervical SCI and upper extremity paralysis whose recovery plateaued were enrolled between September 1, 2015, and January 31, 2019. Data analysis was performed from August 2021 to February 2022. INTERVENTIONS: Nerve transfers to reanimate upper extremity motor function with target reinnervation of elbow extension and hand grasp, pinch, and/or release. MAIN OUTCOMES AND MEASURES: The primary outcome was motor strength measured by Medical Research Council (MRC) grades 0 to 5. Secondary outcomes included Sollerman Hand Function Test (SHFT); Michigan Hand Outcome Questionnaire (MHQ); Disabilities of Arm, Shoulder, and Hand (DASH); and 36-Item Short Form Health Survey (SF-36) physical component summary (PCS) and mental component summary (MCS) scores. Outcomes were assessed up to 48 months postoperatively. RESULTS: Twenty-two patients with tetraplegia (median age, 36 years [range, 18-76 years]; 21 male [95%]) underwent 60 nerve transfers on 35 upper limbs at a median time of 21 months (range, 6-142 months) after SCI. At final follow-up, upper limb motor strength improved significantly: median MRC grades were 3 (IQR, 2.5-4; P = .01) for triceps, with 70% of upper limbs gaining an MRC grade of 3 or higher for elbow extension; 4 (IQR, 2-4; P \u3c .001) for finger extensors, with 79% of hands gaining an MRC grade of 3 or higher for finger extension; and 2 (IQR, 1-3; P \u3c .001) for finger flexors, with 52% of hands gaining an MRC grade of 3 or higher for finger flexion. The secondary outcomes of SHFT, MHQ, DASH, and SF36-PCS scores improved beyond the established minimal clinically important difference. Both early (\u3c12 months) and delayed (≥12 months) nerve transfers after SCI achieved comparable motor outcomes. Continual improvement in motor strength was observed in the finger flexors and extensors across the entire duration of follow-up. CONCLUSIONS AND RELEVANCE: In this prospective case series, nerve transfer surgery was associated with improvement of upper limb motor strength and functional independence in patients with tetraplegia. Nerve transfer is a promising intervention feasible in both subacute and chronic SCI

B844: Checklist of the Vascular Plants of Maine Third Revision

This is the third revision of the Checklist of Vascular Plants of Maine. Like its predecessors, it lists all ferns and related plants, conifers, and flowering plants native and naturalized in Maine and records their county-level distribution in the state. The first Check- list (Ogden et al. 1948) was based on specimens in herbaria at the University of Maine (hereafter referred to as MAINE), Portland Society of Natural History, New England Botanical Club, Gray Herbarium of Harvard University, and the private collection of Glen D. Chamberlain of Presque Isle, Maine (now part of MAINE). Bean et al. (1966) revised the checklist to include additions to the flora and update the nomenclature to follow Fernald (1950). Richards et al. (1983) added many new state and county records in the second revision. The purpose of this revision is twofold. First, we have included many new county and state records. Since Richards et al. (1983) there has been considerable collecting in Maine, much of it directed at searching for new state and county records in relatively neglected regions of the state. Second, there have been numerous changes in the scientific names of Maine plants since Fernald (1950), the nomenclatural basis of Richards et al. (1983). We have largely followed Kartesz\u27s (1994) nomenclature (see Taxonomy and Nomenclature section). Recent work on rare plants and establishment of an official list of endangered and threatened plants in Maine (Dibble et al. 1989; Maine State Planning Office 1990) also motivate updating the known distribution and taxonomy of Maine\u27s flora.https://digitalcommons.library.umaine.edu/aes_bulletin/1121/thumbnail.jp

Elucidating role of salivary proteins in denture stomatitis using a proteomic approach

Denture stomatitis (DS) is the most common oral pathology among denture wearers, affecting over one-third of this group. DS is usually associated with C. albicans. However, unlike other oral candidiasis, most DS patients have intact host immunity. The presence of a denture alone is usually sufficient for DS. Saliva and its protein contents can theoretically predispose some denture wearers to DS and others resistant toward DS. Here we proposed for the first time to define salivary proteomic profiles of denture wearers with and without DS. SELDI-TOF/MS analysis suggests that there is a proteomic differentiation among control, localized and generalized DS. Based on initial SELDI-TOF/MS profiling, we further used reversed phase liquid chromatography, MALDI-TOF/MS, and LC-MS/MS to characterize the salivary proteins associated with DS. Nineteen proteins based on SELDI-TOF/MS profiling were found including cystatin-SN, statherin, kininogen-1, desmocollin-2, carbonic anhydrase-6, peptidyl-prolyl cis–trans isomerase A like peptides, cystatin C, and several immunoglobulin fragments. The proteomic content gives evidence of the interaction between host tissue, saliva, and candida. Further examination in larger populations of these proteins may help to gain a better understanding of DS pathological processes and improve DS treatments

Defining the Functional Domain of Programmed Cell Death 10 through Its Interactions with Phosphatidylinositol-3,4,5-Trisphosphate

Cerebral cavernous malformations (CCM) are vascular abnormalities of the central nervous system predisposing blood vessels to leakage, leading to hemorrhagic stroke. Three genes, Krit1 (CCM1), OSM (CCM2), and PDCD10 (CCM3) are involved in CCM development. PDCD10 binds specifically to PtdIns(3,4,5)P3 and OSM. Using threading analysis and multi-template modeling, we constructed a three-dimensional model of PDCD10. PDCD10 appears to be a six-helical-bundle protein formed by two heptad-repeat-hairpin structures (α1–3 and α4–6) sharing the closest 3D homology with the bacterial phosphate transporter, PhoU. We identified a stretch of five lysines forming an amphipathic helix, a potential PtdIns(3,4,5)P3 binding site, in the α5 helix. We generated a recombinant wild-type (WT) and three PDCD10 mutants that have two (Δ2KA), three (Δ3KA), and five (Δ5KA) K to A mutations. Δ2KA and Δ3KA mutants hypothetically lack binding residues to PtdIns(3,4,5)P3 at the beginning and the end of predicted helix, while Δ5KA completely lacks all predicted binding residues. The WT, Δ2KA, and Δ3KA mutants maintain their binding to PtdIns(3,4,5)P3. Only the Δ5KA abolishes binding to PtdIns(3,4,5)P3. Both Δ5KA and WT show similar secondary and tertiary structures; however, Δ5KA does not bind to OSM. When WT and Δ5KA are co-expressed with membrane-bound constitutively-active PI3 kinase (p110-CAAX), the majority of the WT is co-localized with p110-CAAX at the plasma membrane where PtdIns(3,4,5)P3 is presumably abundant. In contrast, the Δ5KA remains in the cytoplasm and is not present in the plasma membrane. Combining computational modeling and biological data, we propose that the CCM protein complex functions in the PI3K signaling pathway through the interaction between PDCD10 and PtdIns(3,4,5)P3

Rho kinase inhibition rescues the endothelial cell cerebral cavernous malformation phenotype

Cerebral cavernous malformations (CCM) are vascular lesions causing seizures and stroke. Mutations causing inactivation of one of three genes, ccm1, -2, or -3, are sufficient to induce vascular endothelial cell defects resulting in CCM. Herein, we show that loss of expression of the CCM1, -2, or -3 proteins causes a marked increase in expression of the GTPase RhoA. Live cell imaging with a RhoA-specific biosensor demonstrates increased RhoA activity with loss of CCM1, -2, or -3, with an especially pronounced RhoA activation in both the cytosol and the nucleus with loss of CCM1 expression. Increased RhoA activation was associated with Rho kinase-dependent phosphorylation of myosin light chain 2. Functionally, loss of CCM1, -2, or -3 inhibited endothelial cell vessel-like tube formation and extracellular matrix invasion, each of which is rescued by chemical inhibition or short hairpin RNA knockdown of Rho kinase. The findings, for the first time, define a signaling network for CCM1, -2, and -3 in CCM pathology, whereby loss of CCM1, -2, or -3 protein expression results in increased RhoA activity, with the activation of Rho kinase responsible for endothelial cell dysregulation. The results define Rho kinase as a therapeutic target to rescue endothelial cells from loss of CCM protein function. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc

Cerebral cavernous malformation is a vascular disease associated with activated RhoA signaling

Cerebral cavernous malformation (CCM) involves the homozygous inactivating mutations of one of three genes, ccm1, -2, or -3 resulting in hyperpermeable blood vessels in the brain. The CCM1, -2, and -3 proteins form a complex to organize the signaling networks controlling endothelial cell physiology including actin dynamics, tube formation, and adherens junctions. The common biochemical defect with the loss of CCM1, -2, or -3 is increased RhoA activity leading to the activation of Rho-associated coiled coil-forming kinase (ROCK). Inhibition of the ROCK rescues CCM endothelial cell dysfunction, suggesting that the inhibition of RhoA-ROCK signaling may be a therapeutic strategy to prevent or arrest the progression of the CCM lesions