Collagenase treatment decreases muscle stiffness in cerebral palsy: A preclinical ex vivo biomechanical analysis of hip adductor muscle fiber bundles.

AIM: To determine the dose-response relationship of collagenase Clostridium histolyticum (CCH) on collagen content and the change in muscle fiber bundle stiffness after ex vivo treatment of adductor longus biopsies with CCH in children with cerebral palsy (CP). METHOD: Biopsy samples of adductor longus from children with CP (classified in Gross Motor Function Classification System levels IV and V) were treated with 0 U/mL, 200 U/mL, 350 U/mL, or 500 U/mL CCH; percentage collagen reduction was measured to determine the dose-response. Peak and steady-state stresses were determined at 1%, 2.5%, 5%, and 7.5% strain increments; Young's modulus was calculated. RESULTS: Eleven patients were enrolled (nine males, two females, mean age at surgery 6 years 5 months; range: 2-16 years). A linear CCH dose-response relationship was determined. Peak and steady-state stress generation increased linearly at 5.9/2.3mN/mm2 , 12.4/5.3mN/mm2 , 22.2/9.7mN/mm2 , and 33.3/15.5mN/mm2 at each percentage strain increment respectively. After CCH treatment, peak and steady-state stress generation decreased to 3.2/1.2mN/mm2 , 6.5/2.9mN/mm2 , 12.2/5.7mN/mm2 , and 15.4/7.7mN/mm2 respectively (p < 0.004). Young's modulus decreased from 205 kPa to 100 kPa after CCH (p = 0.003). INTERPRETATION: This preclinical ex vivo study provides proof of concept for the use of collagenase to decrease muscle stiffness in individuals with CP.

DNA Methylation Analysis Reveals Distinct Patterns in Satellite Cell-Derived Myogenic Progenitor Cells of Subjects with Spastic Cerebral Palsy.

Spastic type cerebral palsy (CP) is a complex neuromuscular disorder that involves altered skeletal muscle microanatomy and growth, but little is known about the mechanisms contributing to muscle pathophysiology and dysfunction. Traditional genomic approaches have provided limited insight regarding disease onset and severity, but recent epigenomic studies indicate that DNA methylation patterns can be altered in CP. Here, we examined whether a diagnosis of spastic CP is associated with intrinsic DNA methylation differences in myoblasts and myotubes derived from muscle resident stem cell populations (satellite cells; SCs). Twelve subjects were enrolled (6 CP; 6 control) with informed consent/assent. Skeletal muscle biopsies were obtained during orthopedic surgeries, and SCs were isolated and cultured to establish patient-specific myoblast cell lines capable of proliferation and differentiation in culture. DNA methylation analyses indicated significant differences at 525 individual CpG sites in proliferating SC-derived myoblasts (MB) and 1774 CpG sites in differentiating SC-derived myotubes (MT). Of these, 79 CpG sites were common in both culture types. The distribution of differentially methylated 1 Mbp chromosomal segments indicated distinct regional hypo- and hyper-methylation patterns, and significant enrichment of differentially methylated sites on chromosomes 12, 13, 14, 15, 18, and 20. Average methylation load across 2000 bp regions flanking transcriptional start sites was significantly different in 3 genes in MBs, and 10 genes in MTs. SC derived MBs isolated from study participants with spastic CP exhibited fundamental differences in DNA methylation compared to controls at multiple levels of organization that may reveal new targets for studies of mechanisms contributing to muscle dysregulation in spastic CP.

An Emerging Role for Epigenetics in Cerebral Palsy.

Cerebral palsy is a set of common, severe, motor disabilities categorized by a static, nondegenerative encephalopathy arising in the developing brain and associated with deficits in movement, posture, and activity. Spastic CP, which is the most common type, involves high muscle tone and is associated with altered muscle function including poor muscle growth and contracture, increased extracellular matrix deposition, microanatomic disruption, musculoskeletal deformities, weakness, and difficult movement control. These muscle-related manifestations of CP are major causes of progressive debilitation and frequently require intensive surgical and therapeutic intervention to control. Current clinical approaches involve sophisticated consideration of biomechanics, radiologic assessments, and movement analyses, but outcomes remain difficult to predict. There is a need for more precise and personalized approaches involving omics technologies, data science, and advanced analytics. An improved understanding of muscle involvement in spastic CP is needed. Unfortunately, the fundamental mechanisms and molecular pathways contributing to altered muscle function in spastic CP are only partially understood. In this review, we outline evidence supporting the emerging hypothesis that epigenetic phenomena play significant roles in musculoskeletal manifestations of CP.

Transcriptional analysis of muscle tissue and isolated satellite cells in spastic cerebral palsy.

AIM: To analyze transcriptomes from muscle tissue and cells to improve our understanding of differences in gene expression and molecular function in cerebral palsy (CP) muscle. METHOD: In this case-control study, eight participants with CP (five males, three females; mean [SD] age 14y 2mo [1y 8mo]) and 11 comparison individuals (eight males, three females; mean [SD] age 14y 0mo [2y 6mo]) were enrolled after informed consent/assent and skeletal muscle was obtained during surgery. RNA was extracted from tissue and from primary satellite cells grown to form myotubes in vitro. RNA sequencing data were analyzed using validated informatics pipelines. RESULTS: Analysis identified expression of 6308 genes in the tissue samples and 7459 in the cultured cells. Significant differential expression between CP and control was identified in 87 genes in the tissue and 90 genes in isolated satellite cell-derived myotube cultures. INTERPRETATION: Both tissue and cell analyses identified differential expression of genes associated with muscle development and multiple pathways of interest. What this paper adds Expression differences were found in muscle tissue and in isolated muscle cells. There was low variability in expression among cells isolated from different muscles. Expression differences suggest complex functional alterations in spastic cerebral palsy.

Neointimal Hyperplasia after Carotid Transection and Anastomosis Surgery is Associated with Degradation of Decorin and Platelet Derived Growth Factor Signaling.

OBJECTIVE: Intimal hyperplasia (IH) is the expansion of the vascular intimal region after intervention, which can lead to stenosis and eventual failure of vascular grafts or interventional procedures such as angioplasty or stent placement. Our goals were to investigate the development of IH in a rabbit open surgical model and to evaluate the associated pathophysiological processes involving decorin and the platelet derived growth factor-BB / platelet derived growth factor receptor-ß / mitogen activated protein kinase (PDGF/PDGFR-ß/MAPK) pathway. METHODS: We conducted carotid transection and primary anastomosis on five New Zealand White rabbits to induce IH and examined the associated pathophysiological changes. Tissue was obtained for histological and protein analysis on post-operative day 21 using the contralateral vessel as a control. Intimal medial thickness (IMT) was calculated to measure IH and compared with the unoperated side. Western blot analysis was performed on tissue lysates to determine the expression of decorin core protein, PDGF-BB, PDGFR-ß, and phosphorylated-MAPK (ph-MAPK). Immunofluorescence microscopy was used to assess tissue distribution of matrix metalloproteinase-2 (MMP-2) and phosphorylated-PDGFR-ß (ph-PDGFR-ß). RESULTS: Bilateral carotid arteries were harvested on postoperative day 21. We compared the IMT in operated with unoperated specimens. IMT was significantly elevated in operated arteries vs. unoperated arteries in all 5 animals (148.6 µm +/- 9.09 vs. 103.40 µm +/- 7.08; 135.2 µm +/- 8.30 vs. 92.40 µm +/- 2.35; 203.1 µm +/- 30.23 vs.104.00 µm +/- 4.52; 236.2 µm +/- 27.22 vs. 141.50 µm +/- 9.95; 226.9 µm +/- 11.12 vs. 98.8 µm +/- 3.78). Western blot analysis revealed degradation of decorin protein in the operated tissue, including loss of a 50 kDa band and the appearance of a cleaved fragment at 10 kDa. Decorin and MMP-2 were observed, via immunofluorescence microscopy, in the neointima of the operated vessels. Western blot analysis also revealed increased PDGF-BB, PDGFR-ß, and ph-MAPK levels in operated tissue. Immunofluorescent staining for ph-PDGFR-ß primarily localized to the neointima, indicating increased signaling through PDGF in this region. CONCLUSION: Carotid transection and primary reanastomosis in rabbits induced IH that was associated with MMP-2 activation, degradation of decorin, and activation of the PDGF/PDGFR-ß /MAPK pathway. The findings in this study should lead to further mechanistic evaluation of these pathways to better understand the potential to modify the intimal hyperplastic response to surgery.

Human Adventitial Fibroblast Phenotype Depends on the Progression of Changes in Substrate Stiffness.

Adventitial fibroblasts (AFs) are major contributors to vascular remodeling and maladaptive cascades associated with arterial disease, where AFs both contribute to and respond to alterations in their surrounding matrix. The relationships between matrix modulus and human aortic AF (AoAF) function are investigated using poly(ethylene glycol)-based hydrogels designed with matrix metalloproteinase (MMP)-sensitive and integrin-binding peptides. Initial equilibrium shear storage moduli for the substrates examined are 0.33, 1.42, and 2.90 kPa; after 42 days of culture, all hydrogels exhibit similar storage moduli (0.3-0.7 kPa) regardless of initial modulus, with encapsulated AoAFs spreading and proliferating. In 10 and 7.5 wt% hydrogels, modulus decreases monotonically throughout culture; however, in 5 wt% hydrogels, modulus increases after an initial 7 days of culture, accompanied by an increase in myofibroblast transdifferentiation and expression of collagen I and III through day 28. Thereafter, significant reductions in both collagens occur, with increased MMP-9 and decreased tissue inhibitor of metalloproteinase-1/-2 production. Releasing cytoskeletal tension or inhibiting cellular protein secretion in 5 wt% hydrogels block the stiffening of the polymer matrix. Results indicate that encapsulated AoAFs initiate cell-mediated matrix remodeling and demonstrate the utility of dynamic 3D systems to elucidate the complex interactions between cell behavior and substrate properties.

Epigenetic machine learning: utilizing DNA methylation patterns to predict spastic cerebral palsy.

BACKGROUND: Spastic cerebral palsy (CP) is a leading cause of physical disability. Most people with spastic CP are born with it, but early diagnosis is challenging, and no current biomarker platform readily identifies affected individuals. The aim of this study was to evaluate epigenetic profiles as biomarkers for spastic CP. A novel analysis pipeline was employed to assess DNA methylation patterns between peripheral blood cells of adolescent subjects (14.9 ± 0.3 years old) with spastic CP and controls at single CpG site resolution. RESULTS: Significantly hypo- and hyper-methylated CpG sites associated with spastic CP were identified. Nonmetric multidimensional scaling fully discriminated the CP group from the controls. Machine learning based classification modeling indicated a high potential for a diagnostic model, and 252 sets of 40 or fewer CpG sites achieved near-perfect accuracy within our adolescent cohorts. A pilot test on significantly younger subjects (4.0 ± 1.5 years old) identified subjects with 73% accuracy. CONCLUSIONS: Adolescent patients with spastic CP can be distinguished from a non-CP cohort based on DNA methylation patterns in peripheral blood cells. A clinical diagnostic test utilizing a panel of CpG sites may be possible using a simulated classification model. A pilot validation test on patients that were more than 10 years younger than the main adolescent cohorts indicated that distinguishing methylation patterns are present earlier in life. This study is the first to report an epigenetic assay capable of distinguishing a CP cohort.

Reduced arterial elasticity due to surgical skeletonization is ameliorated by abluminal PEG hydrogel.

Arteries for bypass grafting are harvested either with neighboring tissue attached or as skeletonized vessels that are free of surrounding tissue. There are significant benefits to skeletonization, but reports suggest that skeletonized vessels may develop structural defects and are at risk for atherosclerosis. We investigated the specific short-term effects of skeletonization on carotid artery biomechanics and microanatomy in a rabbit model. Six carotid arteries were surgically skeletonized. To support healing, three of these received polyethylene glycol hydrogel injected along their exterior surfaces. M-mode ultrasonography was used to track circumferential cyclic strain in the skeletonized, hydrogel-treated, and contralateral vessels. On day 21, the arteries were harvested, and vessel structure was assessed by histology, immunofluorescence microscopy, two-photon elastin autofluorescence, and second harmonic generation (SHG) microscopy. Intimal-medial thickness appeared unaffected by skeletonization, but the SHG signals indicated significant changes in collagen turnover in the adventitia. Skeletonized arteries also exhibited significantly decreased radial compliance (circumferential cyclic strain dropped ∼30%) and decreased numbers of elastic laminae (9.1 ± 2.0 to 2.3 ± 1.4). Hydrogel treatment protected against these effects with treated vessels maintaining normal mechanical properties. These results indicate that arterial skeletonization triggers immediate effects on vessel remodeling and reduced vessel compliance resulting in specific tissue alterations within 21 days, but that these effects can be attenuated by the placement of hydrogel on the exterior surface of the skeletonized vessel.

Decreasing matrix modulus of PEG hydrogels induces a vascular phenotype in human cord blood stem cells.

Adult and congenital cardiovascular diseases are significant health problems that are often managed using surgery. Bypass grafting is a principal therapy, but grafts fail at high rates due to hyperplasia, fibrosis, and atherosclerosis. Biocompatible, cellularized materials that attenuate these complications and encourage healthy microvascularization could reduce graft failure, but an improved understanding of biomaterial effects on human stem cells is needed to reach clinical utility. Our group investigates stem-cell-loaded biomaterials for placement along the adventitia of at-risk vessels and grafts. Here, the effects of substrate modulus on human CD34+ stem cells from umbilical cord blood were evaluated. Cells were isolated by immunomagnetic separation and encapsulated in 3, 4, and 6 weight% PEG hydrogels containing 0.032% gelatin and 0.0044% fibronectin. Gels reached moduli of 0.34, 4.5, and 9.1 kPa. Cell viability approached 100%. Cell morphologies appeared similar across gels, but proliferation was significantly lower in 6 wt% gels. Expression profiling using stem cell signaling arrays indicated enhanced self-renewal and differentiation into vascular endothelium among cells in the lower weight percent gels. Thus, modulus was associated with cell proliferation and function. Gels with moduli in the low kilopascal range may be useful in stimulating cell engraftment and microvascularization of graft adventitia.

Neuromotor synapses in Escobar syndrome.

The Escobar variant of multiple pterygium syndrome (OMIM #265000) is a rare, autosomal recessive disorder associated with mutations in the γ-subunit of the nicotinic acetylcholine receptor (CHRNG). CHRNG is expressed in fetal muscle during motor development and contributes to the formation of neuromuscular junctions (NMJs). Anomalies in NMJ structure and function have not been investigated in patients with Escobar syndrome. We report five patients identified as having Escobar syndrome, from four families. In three families, the same mutation (c.459dupA) was identified in CHRNG. A biopsy from brachioradialis muscle was collected from a patient from one of these families and analyzed for NMJ organization using fluorescence microscopy. Compared to spinalis muscle from control patients with idiopathic scoliosis or cerebral palsy (CP), the patient with Escobar syndrome had a significantly higher degree of acetylcholine receptor present outside acetylcholinesterase and significantly less acetylcholinesterase outside acetylcholine receptors. Given the role of the acetylcholine receptor γ-subunit in fetal neuromuscular signal transduction and in establishing the primary encounter of muscle and motor nerve terminal, the CHRNG mutations described in Escobar syndrome may cause a broader disruption of postsynaptic proteins and result in aberrant development of the NMJ due to impaired prenatal neuromuscular transmission and/or abnormal neuromuscular synaptogenesis.

Disruption of basal lamina components in neuromotor synapses of children with spastic quadriplegic cerebral palsy.

Cerebral palsy (CP) is a static encephalopathy occurring when a lesion to the developing brain results in disordered movement and posture. Patients present with sometimes overlapping spastic, athetoid/dyskinetic, and ataxic symptoms. Spastic CP, which is characterized by stiff muscles, weakness, and poor motor control, accounts for ∼80% of cases. The detailed mechanisms leading to disordered movement in spastic CP are not completely understood, but clinical experience and recent studies suggest involvement of peripheral motor synapses. For example, it is recognized that CP patients have altered sensitivities to drugs that target neuromuscular junctions (NMJs), and protein localization studies suggest that NMJ microanatomy is disrupted in CP. Since CP originates during maturation, we hypothesized that NMJ disruption in spastic CP is associated with retention of an immature neuromotor phenotype later in life. Scoliosis patients with spastic CP or idiopathic disease were enrolled in a prospective, partially-blinded study to evaluate NMJ organization and neuromotor maturation. The localization of synaptic acetylcholine esterase (AChE) relative to postsynaptic acetylcholine receptor (AChR), synaptic laminin ß2, and presynaptic vesicle protein 2 (SV2) appeared mismatched in the CP samples; whereas, no significant disruption was found between AChR and SV2. These data suggest that pre- and postsynaptic NMJ components in CP children were appropriately distributed even though AChE and laminin ß2 within the synaptic basal lamina appeared disrupted. Follow up electron microscopy indicated that NMJs from CP patients appeared generally mature and similar to controls with some differences present, including deeper postsynaptic folds and reduced presynaptic mitochondria. Analysis of maturational markers, including myosin, syntrophin, myogenin, and AChR subunit expression, and telomere lengths, all indicated similar levels of motor maturation in the two groups. Thus, NMJ disruption in CP was found to principally involve components of the synaptic basal lamina and subtle ultra-structural modifications but appeared unrelated to neuromotor maturational status.

In situ crosslinkable heparin-containing poly(ethylene glycol) hydrogels for sustained anticoagulant release.

Low-molecular weight heparin (LMWH) is widely used in anticoagulation therapies and for the prevention of thrombosis. LMWH is administered by subcutaneous injection usually once or twice per day. This frequent and invasive delivery modality leads to compliance issues for individuals on prolonged therapeutic courses, particularly pediatric patients. Here, we report a long-term delivery method for LMWH via subcutaneous injection of long-lasting hydrogels. LMWH is modified with reactive maleimide groups so that it can be crosslinked into continuous networks with four-arm thiolated poly(ethylene glycol) (PEG-SH). Maleimide-modified LMWH (Mal-LMWH) retains bioactivity as indicated by prolonged coagulation time. Hydrogels comprising PEG-SH and Mal-LMWH degrade via hydrolysis, releasing bioactive LMWH by first-order kinetics with little initial burst release. Separately dissolved Mal-LMWH and PEG-SH solutions were co-injected subcutaneously in New Zealand White rabbits. The injected solutions successfully formed hydrogels in situ and released LMWH as measured via chromogenic assays on plasma samples, with accumulation of LMWH occurring at day 2 and rising to near-therapeutic dose equivalency by day 5. These results demonstrate the feasibility of using LMWH-containing, crosslinked hydrogels for sustained and controlled release of anticoagulants.

Differential effects of substrate modulus on human vascular endothelial, smooth muscle, and fibroblastic cells.

Regenerative medicine approaches offer attractive alternatives to standard vascular reconstruction; however, the biomaterials to be used must have optimal biochemical and mechanical properties. To evaluate the effects of biomaterial properties on vascular cells, heparinized poly(ethylene glycol) (PEG)-based hydrogels of three different moduli, 13.7, 5.2, and 0.3 kPa, containing fibronectin and growth factor were utilized to support the growth of three human vascular cell types. The cell types exhibited differences in attachment, proliferation, and gene expression profiles associated with the hydrogel modulus. Human vascular smooth muscle cells demonstrated preferential attachment on the highest-modulus hydrogel, adventitial fibroblasts demonstrated preferential growth on the highest-modulus hydrogel, and human umbilical vein endothelial cells demonstrated preferential growth on the lowest-modulus hydrogel investigated. Our studies suggest that the growth of multiple vascular cell types can be supported by PEG hydrogels and that different populations can be controlled by altering the mechanical properties of biomaterials.

Three-dimensional culture alters primary cardiac cell phenotype.

The directed formation of complex three-dimensional (3D) tissue architecture is a fundamental goal in tissue engineering and regenerative medicine. The growth of cells in 3D structures is expected to influence cellular phenotype and function, especially relative cell distribution, expression profiles, and responsiveness to exogenous signals; however, relatively few studies have been carried out to examine the effects of 3D reaggregation on cells from critical target organs, like the heart. Accordingly, we cultured primary cardiac ventricular cells in a 3D model system using a serum-free medium to test the hypothesis that expression profiles, multicellular organizational pathways, tissue maturation markers, and responsiveness to hormone stimulation were significantly altered in stable cell populations grown in 3D versus 2D culture. We found that distinct multi-cellular structures formed in 3D in conjunction with changes in mRNA expression profile, up-regulation of endothelial cell migratory pathways, decreases in the expression of fetal genes (Nppa and Ankrd1), and increased sensitivity to tri-iodothyronine stimulation when compared to parallel 2D cultures comprising the same cell populations. These results indicate that the culture of primary cardiac cells in 3D aggregates leads to physiologically relevant alterations in component cell phenotype consistent with cardiac ventricular tissue formation and maturation.

Robust quantification of the SMN gene copy number by real-time TaqMan PCR.

Spinal muscular atrophy (SMA) is an autosomal recessive disease caused by mutation or deletion of the survival motor neuron gene 1 (SMN1). The highly homologous gene, SMN2, is present in all patients, but it cannot compensate for loss of SMN1. SMN2 differs from SMN1 by a few nucleotide changes, but a C --> T transition in exon 7 leads to exon skipping. As a result, most transcripts from the SMN2 gene lack exon 7. Although SMN1 is the disease-determining gene, the number of SMN2 copies appears to modulate SMA clinical phenotypes. Thus, determining the SMN copy number is important for clinical diagnosis and prognosis. We have developed a quantitative real-time TaqMan polymerase chain reaction assay for both the SMN1 and SMN2 genes, in which reliable copy number determination was possible on deoxyribonucleic acid samples obtained by two different isolation methods and from two different sources (human blood and skin fibroblasts). For SMN1, allele specificity was attained solely by addition of an allele-specific forward primer and, for SMN2, by addition of a specific forward primer and a nonextending oligonucleotide (SMN1 blocker) that reduced nonspecific amplification from SMN1 to a negligible level. We validated the reliability of this real-time polymerase chain reaction approach and found that the coefficient of variation for all the gene copy number measurements was below 10%. Quantitative analysis of the SMN copy number in SMA fibroblasts by this approach showed deletion of SMN1 and an inverse correlation between the SMN2 copy number and severity of the disease.