Factors Associated with Antihypertensive Therapy Prescription in Youth Delaware Medicaid Recipients with Primary Hypertension Diagnosis.

BACKGROUND: Higher neighborhood deprivation is associated with hypertension diagnosis in youth. In this study, we assess if there is an association between neighborhood deprivation and antihypertensive therapy prescription among insured youth with a primary hypertension diagnosis. METHODS: Using a retrospective cross-sectional design, we assessed the proportion of youth with a diagnosis of primary hypertension prescribed antihypertensive therapy. We evaluated the proportion of youth prescribed antihypertensive therapy and compared prescribing patterns by area deprivation index (ADI), age, sex, obesity diagnosis, race, ethnicity, and duration of Medicaid coverage. RESULTS: Of the 65,452 non-pregnant Delaware Medicaid recipients, 8-18 years of age, 1,145 (1.7%) had an International classification of diseases (ICD)-9/ICD-10 diagnosis of primary hypertension; 165 of the 1,145 (14%) were prescribed antihypertensive therapy. Factors associated with a greater odds of prescription by multivariable logistic regression were age, obesity diagnosis, and duration of Medicaid full benefit coverage. Odds of antihypertensive therapy prescription did not vary by race, ethnicity, or ADI. CONCLUSIONS: Antihypertensive therapy prescription rates are poor despite national guideline recommendations. Among youth receiving Delaware Medicaid between 2014 and 2019, prescription proportions were highest among youth of older age, with an obesity diagnosis, and among youth with longer duration of Medicaid benefit coverage. Although high area deprivation has been shown to be associated with the diagnosis of hypertension, high vs. low area deprivation was not associated with greater antihypertensive therapy prescription among youth with primary hypertension. Our finding of a mismatch between hypertension diagnosis and antihypertensive therapy prescription highlights a potential disparity in antihypertensive therapy prescription in youth.

Exploring the Multifaceted Biologically Relevant Roles of circRNAs: From Regulation, Translation to Biomarkers.

CircRNAs are a category of regulatory RNAs that have garnered significant attention in the field of regulatory RNA research due to their structural stability and tissue-specific expression. Their circular configuration, formed via back-splicing, results in a covalently closed structure that exhibits greater resistance to exonucleases compared to linear RNAs. The distinctive regulation of circRNAs is closely associated with several physiological processes, as well as the advancement of pathophysiological processes in several human diseases. Despite a good understanding of the biogenesis of circular RNA, details of their biological roles are still being explored. With the steady rise in the number of investigations being carried out regarding the involvement of circRNAs in various regulatory pathways, understanding the biological and clinical relevance of circRNA-mediated regulation has become challenging. Given the vast landscape of circRNA research in the development of the heart and vasculature, we evaluated cardiovascular system research as a model to critically review the state-of-the-art understanding of the biologically relevant functions of circRNAs. We conclude the review with a discussion of the limitations of current functional studies and provide potential solutions by which these limitations can be addressed to identify and validate the meaningful and impactful functions of circRNAs in different physiological processes and diseases.

The Typical tRNA Co-Expresses Multiple 5' tRNA Halves Whose Sequences and Abundances Depend on Isodecoder and Isoacceptor and Change with Tissue Type, Cell Type, and Disease.

Transfer RNA-derived fragments (tRFs) are noncoding RNAs that arise from either mature transfer RNAs (tRNAs) or their precursors. One important category of tRFs comprises the tRNA halves, which are generated through cleavage at the anticodon. A given tRNA typically gives rise to several co-expressed 5'-tRNA halves (5'-tRHs) that differ in the location of their 3' ends. These 5'-tRHs, even though distinct, have traditionally been treated as indistinguishable from one another due to their near-identical sequences and lengths. We focused on co-expressed 5'-tRHs that arise from the same tRNA and systematically examined their exact sequences and abundances across 10 different human tissues. To this end, we manually curated and analyzed several hundred human RNA-seq datasets from NCBI's Sequence Run Archive (SRA). We grouped datasets from the same tissue into their own collection and examined each group separately. We found that a given tRNA produces different groups of co-expressed 5'-tRHs in different tissues, different cell lines, and different diseases. Importantly, the co-expressed 5'-tRHs differ in their sequences, absolute abundances, and relative abundances, even among tRNAs with near-identical sequences from the same isodecoder or isoacceptor group. The findings suggest that co-expressed 5'-tRHs that are produced from the same tRNA or closely related tRNAs have distinct, context-dependent roles. Moreover, our analyses show that cell lines modeling the same tissue type and disease may not be interchangeable when it comes to experimenting with tRFs.

Launching IDeA state early career clinician-scientists with mentored just-in-time grant-writing support.

Institutional Development Award (IDeA) programs build research infrastructure in regions with historically low access to NIH funds. The Mentored Research Development Award (MRDA), a professional development program embedded in our IDeA-funded center, provides junior investigators with mentorship and effort offset to write a grant. We evaluated outcomes from the first eight years (2013-2021; N = 55) using administrative records, publicly available data, and a self-report survey (n = 46, 84% response rate). Fifteen MRDA recipients (27%) went on to receive NIH funding. Providing just-in-time grant-writing support may launch early career clinician-scientists in an IDeA state context.

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.

Association of Area Deprivation With Primary Hypertension Diagnosis Among Youth Medicaid Recipients in Delaware.

Importance: The association between degree of neighborhood deprivation and primary hypertension diagnosis in youth remains understudied. Objective: To assess the association between neighborhood measures of deprivation and primary hypertension diagnosis in youth. Design, Setting, and Participants: This cross-sectional study included 65 452 Delaware Medicaid-insured youths aged 8 to 18 years between January 1, 2014, and December 31, 2019. Residence was geocoded by national area deprivation index (ADI). Exposures: Higher area deprivation. Main Outcomes and Measures: The main outcome was primary hypertension diagnosis based on International Classification of Diseases, Ninth Revision and Tenth Revision codes. Data were analyzed between September 1, 2021, and December 31, 2022. Results: A total of 65 452 youths were included in the analysis, including 64 307 (98.3%) without a hypertension diagnosis (30 491 [47%] female and 33 813 [53%] male; mean [SD] age, 12.5 (3.1) years; 12 500 [19%] Hispanic, 25 473 [40%] non-Hispanic Black, 24 565 [38%] non-Hispanic White, and 1769 [3%] other race or ethnicity; 13 029 [20%] with obesity; and 31 548 [49%] with an ADI ≥50) and 1145 (1.7%) with a diagnosis of primary hypertension (mean [SD] age, 13.3 [2.8] years; 464 [41%] female and 681 [59%] male; 271 [24%] Hispanic, 460 [40%] non-Hispanic Black, 396 [35%] non-Hispanic White, and 18 [2%] of other race or ethnicity; 705 [62%] with obesity; and 614 [54%] with an ADI ≥50). The mean (SD) duration of full Medicaid benefit coverage was 61 (16) months for those with a diagnosis of primary hypertension and 46.0 (24.3) months for those without. By multivariable logistic regression, residence within communities with ADI greater than or equal to 50 was associated with 60% greater odds of a hypertension diagnosis (odds ratio [OR], 1.61; 95% CI 1.04-2.51). Older age (OR per year, 1.16; 95%, CI, 1.14-1.18), an obesity diagnosis (OR, 5.16; 95% CI, 4.54-5.85), and longer duration of full Medicaid benefit coverage (OR, 1.03; 95% CI, 1.03-1.04) were associated with greater odds of primary hypertension diagnosis, whereas female sex was associated with lower odds (OR, 0.68; 95%, 0.61-0.77). Model fit including a Medicaid-by-ADI interaction term was significant for the interaction and revealed slightly greater odds of hypertension diagnosis for youths with ADI less than 50 (OR, 1.03; 95% CI, 1.03-1.04) vs ADI ≥50 (OR, 1.02; 95% CI, 1.02-1.03). Race and ethnicity were not associated with primary hypertension diagnosis. Conclusions and Relevance: In this cross-sectional study, higher childhood neighborhood ADI, obesity, age, sex, and duration of Medicaid benefit coverage were associated with a primary hypertension diagnosis in youth. Screening algorithms and national guidelines may consider the importance of ADI when assessing for the presence and prevalence of primary hypertension in youth.

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.

Association of key species of vaginal bacteria of recurrent bacterial vaginosis patients before and after oral metronidazole therapy with short- and long-term clinical outcomes.

Bacterial vaginosis (BV) is associated with a state of vaginal dysbiosis typically involving depletion of otherwise dominant populations of Lactobacillus. The causes of this microbial succession are not known; there may be multiple causes. Standard treatment includes oral metronidazole, which typically restores Lactobacillus species to dominance. However, recurrence rates are high; recurrent BV patients recur 3-4 times annually and are often refractory to treatment. Our previous qPCR-based study of recurrent BV patients pointed to putatively more virulent species of Gardnerella that were associated with refractory responses to oral metronidazole, and less robust recovery of Lactobacillus species associated with recurrence after an initial period of remission. However, these associations did not account for outcomes in all patients, suggesting that other bacterial species were involved. In this follow-up study, we sequenced the V4 domain of 16S rRNA sequences of 41of these same patients pre- and posttreatment. Overall compositions among pretreatment clinical outcome groups were not different, although alpha diversity significantly decreased: refractory > recurrent > remission. Combinations of key species were associated with and prognostic for outcome. Higher pretreatment abundance of Megasphaera lornae together with lower abundance of Gardnerella Gsp07 and Finegoldia magna predicted long term remission after oral metronidazole. Furthermore, a subset of refractory patients that did not have high levels of Gardnerella Gsp07, instead had elevated levels of alternative species including Atopobium vaginae, Mageeibacillus indolicus (BVAB3), and Prevotella timonensis. Patients who recurred after transient remission had elevated abundance of species including Atopobium vaginae, Gardnerella, and Aerococcus christensenii, compared to long-term remission patients. Core bacterial species among refractory patients did not change in abundance after metronidazole, suggesting resistance or tolerance, in contrast to the loss in abundance of the same species among recurrent or remission patients. These findings have potential prognostic and therapeutic implications.

Determining Susceptibility in Candida Vaginal Isolates.

Antifungal drug susceptibility tests (AST) for Candida albicans are increasingly demanded for women with refractory or recurrent Candida vaginitis due to fluconazole resistance. Given reduced activity of azole drugs at pH levels found in women with Candida vaginitis, it is proposed that AST be performed at pH 4.5, since testing at only the recommended pH 7.0 is likely to miss a significant number of clinically relevant azole-resistant C. albicans vaginal isolates.

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.

Prognosis of recurrent bacterial vaginosis based on longitudinal changes in abundance of Lactobacillus and specific species of Gardnerella.

Refractory responses to standard-of-care oral metronidazole among recurrent bacterial vaginosis (BV) patients is not rare, and recurrence within a year is common. A better understanding of the bacterial determinants of these outcomes is essential. In this study we ask whether changes in specific species of Gardnerella are associated with poor short or long term clinical outcomes, and if and how resurgence of Lactobacillus species affects these outcomes. We quantify Lactobacillus isolates as a proportion of total vaginal bacteria using the LbRC5 qPCR assay, and 5 prevalent species of Gardnerella using primers that target species-specific polymorphisms within the cpn60 gene. The study includes 43 BV patients: 18 refractory, 16 recurrent, and 11 remission patients, sampled daily for up to two weeks post-treatment; clinical outcomes were tracked for up to 9 months. Persistently high titers of Gardnerella Gsp07 were associated with refractory responses, and persistently low abundance of Gardnerella Gsp07 and G. swidsinskii / G. leopoldii were associated with remission. Lactobacillus species abundance rose in 4-14 days after initiation of treatment in most but not all recurrent and remission patients, although increases were more sustained among remission patients. The findings suggest that Gardnerella Gsp07 and G. swidsinskii / G. leopoldii are markers of poor clinical outcome or may directly or indirectly suppress recovery of Lactobacillus species, thereby interfering with clinical recovery. Therapies that target these strains may improve patient outcome.

Resistance to Neuromuscular Blockade by Rocuronium in Surgical Patients with Spastic Cerebral Palsy.

Individuals with spastic cerebral palsy (CP) often exhibit altered sensitivities to neuromuscular blocking agents (NMBAs) used for surgical intubation. We assessed usage of the NMBA rocuronium in patients with spastic CP and evaluated potential modifiers of dosing including gross motor function classification system (GMFCS) level, birthweight, gestational age, and the use of anticonvulsant therapy. In a case-control study, surgical patients with spastic CP (n = 64) or with idiopathic or non-neuromuscular conditions (n = 73) were enrolled after informed consent/assent. Patient data, GMFCS level, anticonvulsant use, and rocuronium dosing for intubation and post-intubation neuromuscular blockade were obtained from medical records. Findings reveal participants with CP required more rocuronium per body weight for intubation than controls (1.00 ± 0.08 versus 0.64 ± 0.03 mg/kg; p < 0.0001). Dosing increased with GMFCS level (Spearman's rho = 0.323; p = 0.005), and participants with moderate to severe disability (GMFCS III-V) had elevated rocuronium with (1.21 ± 0.13 mg/kg) or without (0.86 ± 0.09 mg/kg) concurrent anticonvulsant therapy. Children born full-term or with birthweight >2.5 kg in the CP cohort required more rocuronium than preterm and low birthweight counterparts. Individuals with CP exhibited highly varied and significant resistance to neuromuscular blockade with rocuronium that was related to GMFCS and gestational age and weight at birth.

Recurrent Bacterial Vaginosis: An Unmet Therapeutic Challenge. Experience With a Combination Pharmacotherapy Long-Term Suppressive Regimen.

BACKGROUND: Recurrence of bacterial vaginosis (RBV) is a major challenge to effective therapy. Women experiencing intractable and frequent recurrences are ill-served by available treatment options, such as both antimicrobial and use of probiotics. METHODS: One hundred five women with RBV failing all recommended regimens seen in the clinic were prescribed combination oral nitroimidazole 500 mg twice a day for 7 days and simultaneous boric acid 600 mg daily per vagina therapy for 30 days; thereafter, they were prescribed twice-weekly vaginal metronidazole gel for 5 months in an attempt to prevent recurrence and followed by a 6-month observation period. Results reflect standard of clinic care in this uncontrolled retrospective cohort analysis. RESULTS: An initial regimen of nitroimidazole and simultaneous but prolonged vaginal boric acid achieved a satisfactory response (BV cure ≤2 Amsel criteria) in 92 of 93 available patients. Thereafter, a maintenance metronidazole gel prevented symptomatic BV recurrence in 69.6% of compliant patients at 6-month follow-up. Long-term cure at a 12-month follow-up was demonstrated in almost 69% of women reaching the 6-month observation phase. Vaginal candidiasis frequently complicated prolonged antibiotic prophylaxis requiring frequent antifungal rescue or prophylaxis. Frequent loss to follow-up in this long-term study influenced efficacy evaluation. CONCLUSIONS: In the absence of new antimicrobials or proven probiotic regimens, women with RBV may benefit from a prolonged drug-intensive antimicrobial regimen incorporating antibiofilm activity until newer measures are available. Additional randomized, control studies are needed.

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.

Substrate stiffness directs the phenotype and polarization state of cord blood derived macrophages.

Cord blood (CB) mononuclear cell populations have demonstrated significant promise in biomaterials-based regenerative therapies; however, the contributions of monocyte and macrophage subpopulations towards proper tissue healing and regeneration are not well understood, and the phenotypic responses of macrophage to microenvironmental cues have not been well-studied. In this work, we evaluated the effects of cytokine stimulation and altered substrate stiffness. Macrophage derived from CB CD14+ monocytes adopted distinct inflammatory (M1) and anti-inflammatory (M2a and M2c) phenotypes in response to cytokine stimulation (M1: lipopolysaccharide (LPS) and interferon (IFN-γ); M2a: interleukin (IL)-4 and IL-13; M2c: IL-10) as determined through expression of relevant cell surface markers and growth factors. Cytokine-induced macrophage readily altered their phenotypes upon sequential administration of different cytokine cocktails. The impact of substrate stiffness on macrophage phenotype was evaluated by seeding CB-derived macrophage on 3wt%, 6wt%, and 14wt% poly(ethylene glycol)-based hydrogels, which exhibited swollen shear moduli of 0.1, 3.4, and 10.3 kPa, respectively. Surface marker expression and cytokine production varied depending on modulus, with anti-inflammatory phenotypes increasing with elevated substrate stiffness. Integration of specific hydrogel moduli and cytokine cocktail treatments resulted in the differential regulation of macrophage phenotypic biomarkers. These data suggest that CB-derived macrophages exhibit predictable behaviors that can be directed and finely tuned by combinatorial modulation of substrate physical properties and cytokine profiles.

Regulation of neovasculogenesis in co-cultures of aortic adventitial fibroblasts and microvascular endothelial cells by cell-cell interactions and TGF-ß/ALK5 signaling.

Adventitial fibroblasts (AFs) are critical mediators of vascular remodeling. However, the contributions of AFs towards development of vasculature and the specific mechanisms by which these cells regulate physiological expansion of the vasa vasorum, the specialized microvasculature that supplies nutrients to the vascular wall, are not well understood. To determine the regulatory role of AFs in microvascular endothelial cell (MVEC) neovasculogenesis and to investigate the regulatory pathways utilized for communication between the two cell types, AFs and MVECs were cultured together in poly(ethylene glycol)-based hydrogels. Following preliminary evaluation of a set of cell adhesion peptides (AG10, AG73, A2G78, YIGSR, RGD), 7.5wt% hydrogels containing 3 mM RGD were selected as these substrates did not initiate primitive tubule structures in 3D MVEC monocultures, thus providing a passive platform to study AF-MVEC interaction. The addition of AFs to hydrogels promoted MVEC viability; however, increasing AF density within hydrogels stimulated MVEC proliferation, increased microvessel density and size, and enhanced deposition of basement membrane proteins, collagen IV and laminin. Importantly, AF-MVEC communication through the transforming growth factor beta (TGF-ß)/activin receptor-like kinase 5 (ALK5) signaling pathway was observed to mediate microvessel formation, as inhibition of ALK5 significantly decreased MVEC proliferation, microvessel formation, mural cell recruitment, and basement membrane production. These data indicate that AFs regulate MVEC neovasculogenesis and suggest that therapeutics targeting the TGF-ß/ALK5 pathway may be useful for regulation of vasculogenic and anti-vasculogenic responses.

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.

Conventional oral and secondary high dose vaginal metronidazole therapy for recurrent bacterial vaginosis: clinical outcomes, impacts of sex and menses.

PURPOSE: Oral metronidazole therapy is the standard of care for bacterial vaginosis (BV), yet it has alarming rates of recurrence and refractory responses among recurrent BV (RBV) patients. This study addresses whether high dose vaginal metronidazole therapy (HDM) is beneficial in RBV patients who fail after standard of care (SOC) therapy, whether diagnostic test scores proximal to the HDM predict clinical outcome, and whether menses, coitus, or race influences therapy outcome. PATIENTS AND METHODS: A total of 90 patients with RBV were given SOC and tracked 74 for up to 9 months. Refractory or recurrent patients (57) with symptomatic BV were given HDM and followed for up to 8 months. Patients were evaluated by Amsel criteria, Nugent score, and a qPCR assay that assesses the Lactobacillus content. RESULTS: HDM achieved at least short-term remission in 68% of the patients who were refractory to or recurred after SOC and provided a 10-day increase in the mean duration of remission among patients who eventually recurred (p=0.027). Patients with prolonged dysbiosis (pH >5 or Amsel 4) before symptomatic recurrence were more likely to recur after subsequent HDM. Most recurrence happened within 10 days of menses, but sex in this cohort was not associated with clinical outcome. Mean diagnostic BV scores of African American patients in remission were inferior to scores of a small cohort of Caucasian patients in remission. CONCLUSION: Encouraging results obtained with HDM justify a prospective, randomized study to determine if follow-up HDM is beneficial among a broader cohort of women failing conventional oral metronidazole therapy.

Spatial Patterning of Molecular Cues and Vascular Cells in Fully Integrated Hydrogel Channels via Interfacial Bioorthogonal Cross-Linking.

Fully integrated hydrogel channels were fabricated via interfacial bioorthogonal cross-linking, a diffusion-controlled method for the creation and patterning of synthetic matrices based on the rapid bioorthogonal reaction between s-tetrazines (Tz) and trans-cyclooctene (TCO) dienophiles. Injecting an aqueous solution of a bisTCO cross-linker into a reservoir of tetrazine-modified hyaluronic acid (HA-Tz), while simultaneously drawing the syringe needle through the reservoir, yielded a cross-linked hydrogel channel that was mechanically robust. Fluorescent tags and biochemical signals were spatially patterned into the channel wall through time-dependent perfusion of TCO-conjugated molecules into the lumen of the channel. Different cell populations were spatially encapsulated in the channel wall via temporal alteration of cells in the HA-Tz reservoir. The interfacial approach enabled the spatial patterning of vascular cells, including human abdominal aorta endothelial cells, aortic vascular smooth muscle cells, and aortic adventitial fibroblasts, into the hydrogel channels with high viability and proper morphology in the anatomical order found in human arteries. The bioorthogonal platform does not rely on external triggers and represents the first step toward the engineering of functional and implantable arteries.