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Due to their physiological relevance, multicellular 3D spheroids are actively replacing standard 2D monolayer cultures. How spheroids are formed through the assembly of individual cells in natural fibrous environments that include a mix of diameters and architectures in vivo remains unknown. Here, we demonstrate that the spontaneous assembly of human vasa vasorum-derived pericytes in 3D spheroids depends on the fiber diameter and network architecture. A parallel arrangement of suspended fibers of all tested diameters (200, 500, and 800 nm) leads to the formation of spheroids, while on crosshatch networks, spheroid assembly on larger diameters is absent. The design of fibrous networks of a mix of diameters and architectures leads to the patterning of spheroids in desired locations. Fiber remodeling in parallel arrangements serves as force sensors providing mechanical insights into the assembly dynamics of spheroids and subsequent cell sprouting from spheroids. Translocation and merger of spheroids occur predominantly on parallel fiber networks, while on crosshatch networks, a cellular exchange is observed between spheroids connected with remodeled fibers. Rho kinase inhibition by Y27632 and subsequent wash-off leads to spheroid disintegration and reassembly, thus, highlighting the role of cell contractility in the assembly and integrity of 3D spheroids. Overall, using extracellular mimicking fiber networks of varying diameters and architectures, we report new insights into the 3D dynamics of spheroids which may inform pericyte’s role in vasculogenesis, and (patho)physiological angiogenesis

Not all academic librarians who oversee children’s collections for pre-service teachers have backgrounds in K-12 education. To develop a robust collection, the St. Cloud State University library re...

Summary Bicuspid aortic valve (BAV) with ∼1%–2% prevalence is the most common congenital heart defect (CHD). It frequently results in valve disease and aorta dilation and is a major cause of adult cardiac surgery. BAV is genetically linked to rare left-heart obstructions (left ventricular outflow tract obstructions [LVOTOs]), including hypoplastic left heart syndrome (HLHS) and coarctation of the aorta (CoA). Mouse and human studies indicate LVOTO is genetically heterogeneous with a complex genetic etiology. Homozygous mutation in the Pcdha protocadherin gene cluster in mice can cause BAV, and also HLHS and other LVOTO phenotypes when accompanied by a second mutation. Here we show two common deletion copy number variants (delCNVs) within the PCDHA gene cluster are associated with LVOTO. Analysis of 1,218 white individuals with LVOTO versus 463 disease-free local control individuals yielded odds ratios (ORs) at 1.47 (95% confidence interval [CI], 1.13–1.92; p = 4.2 × 10−3) for LVOTO, 1.47 (95% CI, 1.10–1.97; p = 0.01) for BAV, 6.13 (95% CI, 2.75–13.7; p = 9.7 × 10−6) for CoA, and 1.49 (95% CI, 1.07–2.08; p = 0.019) for HLHS. Increased OR was observed for all LVOTO phenotypes in homozygous or compound heterozygous PCDHA delCNV genotype comparison versus wild type. Analysis of an independent white cohort (381 affected individuals, 1,352 control individuals) replicated the PCDHA delCNV association with LVOTO. Generalizability of these findings is suggested by similar observations in Black and Chinese individuals with LVOTO. Analysis of Pcdha mutant mice showed reduced PCDHA expression at regions of cell-cell contact in aortic smooth muscle and cushion mesenchyme, suggesting potential mechanisms for BAV pathogenesis and aortopathy. Together, these findings indicate common variants causing PCDHA deficiency play a significant role in the genetic etiology of common and rare LVOTO-CHD.

OBJECTIVES We hypothesized that expression and activity of nitric oxide synthase-3 enzyme (Nos3) in bicuspid aortic valve (BAV) aortopathy are related to tissue layer and Nos3 genotype. METHODS Gene expression of Nos3 and platelet and endothelial cell adhesion molecule-1 (Pecam1) and NOS activity were measured in intima-containing media and adventitial specimens of ascending aortic tissue. The presence of 2 Nos3 single-nucleotide polymorphisms (SNPs; −786T/C and 894G/T) was determined for non-aneurysmal (NA) and aneurysmal patients with BAV (n = 40, 89, respectively); patients with tricuspid aortic valve (TAV) and aneurysm (n = 151); and NA patients with TAV (n = 100). RESULTS Elevated Nos3 relative to Pecam1 and reduced Pecam1 relative to a housekeeping gene were observed within intima-containing aortic specimens from BAV patients when compared with TAV patients. Lower Nos3 in the adventitia of aneurysmal specimens was noted when compared with specimens of NA aorta, independent of valve morphology. NOS activity was similar among cohorts in media/intima and decreased in the diseased adventitia, relative to control patients. Aneurysmal BAV patients exhibited an under-representation of the wild-type genotype for −786 SNP. No differences in genotype distribution were noted for 894 SNP. Primary intimal endothelial cells from patients with at least 1 C allele at −786 SNP exhibited lower Nos3 when compared with wild-type cells. CONCLUSIONS These findings of differential Nos3 in media/intima versus adventitia depending on valve morphology or aneurysm reveal new information regarding aneurysmal pathophysiology and support our ongoing assertion that there are distinct mechanisms giving rise to ascending aortopathy in BAV and TAV patients.

Background Homelessness is associated with substantial morbidity. Data linkages between homeless and health systems are important to understand unique needs across homeless populations, identify homeless individuals not registered in homeless databases, quantify the impact of housing services on health-system use, and motivate health systems and payers to contribute to housing solutions. Methods We performed a cross-sectional survey including six health systems and two Homeless Management Information Systems (HMIS) in Cook County, Illinois. We performed privacy-preserving record linkage to identify homelessness through HMIS or ICD-10 codes captured in electronic medical records. We measured the prevalence of health conditions and health-services use across the following typologies: housing-service utilizers stratified by service provided (stable, stable plus unstable, unstable) and non-utilizers (i.e., homelessness identified through diagnosis codes—without receipt of housing services). Results Among 11,447 homeless recipients of healthcare, nearly 1 in 5 were identified by ICD10 code alone without recorded homeless services (n = 2177; 19%). Almost half received homeless services that did not include stable housing (n = 5444; 48%), followed by stable housing (n = 3017; 26%), then receipt of both stable and unstable services (n = 809; 7%). Setting stable housing recipients as the referent group, we found a stepwise increase in behavioral-health conditions from stable housing to those known as homeless solely by health systems. Compared to those in stable housing, prevalence rate ratios (PRR) for those without homeless services were as follows: depression (PRR = 2.2; 95% CI 1.9 to 2.5), anxiety (PRR = 2.5; 95% CI 2.1 to 3.0), schizophrenia (PRR = 3.3; 95% CI 2.7 to 4.0), and alcohol-use disorder (PRR = 4.4; 95% CI 3.6 to 5.3). Homeless individuals who had not received housing services relied on emergency departments for healthcare—nearly 3 of 4 visited at least one and many (24%) visited multiple. Conclusions Differences in behavioral-health conditions and health-system use across homeless typologies highlight the particularly high burden among homeless who are disconnected from homeless services. Fragmented and high use of emergency departments for care should motivate health systems and payers to promote housing solutions, especially those that incorporate substance use and mental health treatment.

Summary In the microcirculation, pericytes are believed to function as mesenchymal stromal cells (MSCs). We hypothesized that the vasa vasorum harbor progenitor cells within the adventitia of human aorta. Pericytes, endothelial progenitor cells, and other cell subpopulations were detected among freshly isolated adventitial cells using flow cytometry. Purified cultured pericytes were enriched for the MSC markers CD105 and CD73 and depleted of the endothelial markers von Willebrand factor and CD31. Cultured pericytes were capable of smooth muscle lineage progression including inducible expression of smooth muscle myosin heavy chain, calponin, and α-smooth muscle actin, and adopted a spindle shape. Pericytes formed spheroids when cultured on Matrigel substrates and peripherally localized with branching endothelial cells in vitro. Our results indicate that the vasa vasorum form a progenitor cell niche distinct from other previously described progenitor populations in human adventitia. These findings could have important implications for understanding the complex pathophysiology of human aortic disease., Highlights • Perivascular progenitor cells were classified in human ascending aorta • Adventitial vasa vasorum were identified as a progenitor cell niche • Purified pericytes were functional in vitro as smooth muscle cell progenitors • Branching endothelial cell networks were associated with pericytes in vitro, Phillippi and colleagues classified the surface proteome of perivascular progenitor cells in human ascending aorta and revealed their localization to the vasa vasorum. Purified pericytes demonstrated potential for smooth muscle cell differentiation and an association with branching endothelial cells in vitro.

Objective Congenital bicuspid aortic valve (BAV) is distinctly associated with the development of ascending aortopathy in adulthood, portending risk of both ascending aortic aneurysm and dissection. Our previous work implicated deficiency in oxidative stress response as a mediator of the BAV-associated aortopathy. We hypothesize that reactive oxygen species generation invokes elevated local oxidative tissue damage in ascending aorta of patients with BAV. Methods Ascending aortic specimens were obtained from patients undergoing elective aortic replacement and/or aortic valve replacement and during heart transplant operations. Levels of superoxide anion were measured via high-pressure liquid chromatography–based detection of 2-hydroxyethidium in aortic specimens. Lipid peroxidation and enzymatic activity of superoxide dismutase and peroxidase were quantified in aortic specimens. Results Superoxide anion production was elevated in aortic specimens from patients with nonaneurysmal BAV (n = 59) compared with specimens from patients with the morphologically normal tricuspid aortic valve (TAV, n = 38). Total superoxide dismutase activity was similar among aortic specimens from patients with TAV versus BAV (n = 27 and 26, respectively), whereas peroxidase activity was increased in aortic specimens from patients with BAV compared with specimens from patients with TAV (n = 14 for both groups). Lipid peroxidation was elevated in aortic specimens from BAV patients compared with TAV patients (n = 14 and 11, respectively). Conclusions Superoxide anion accumulation and increased lipid peroxidation demonstrate that, despite increased peroxidase activity, the ascending aortopathy of patients with BAV involves oxidative stress. In addition, the absence of increased superoxide dismutase activity in BAV specimens indicates a deficiency in antioxidant defense. This suggests that the characteristic smooth muscle cell loss observed in BAV aortopathy may be a consequence of superoxide-mediated cell damage.

Assessing library resources and services at a distance holds unique challenges in gathering data needed to make informed decisions. This article describes the complete process of piloting virtual focus groups—from planning and implementation through the analysis of results for a completely online student population. The virtual focus group method proved effective in getting qualitative feedback to spur library improvements, and it is transferable to many different library settings.

International audience; BACKGROUND: Endothelial nitric oxide (NO) synthase (eNOS) has been implicated in the development of bicuspid aortic valve (BAV) and with differential expression in the ascending aorta of BAV patients. However, little is known about functional disruptions in the eNOS pathway and the effect on BAV-associated aortic dilatation. We tested the hypothesis that eNOS function is regionally diminished in ascending thoracic aortic aneurysms associated with BAV. METHODS: Thoracic aortic aneurysms specimens were collected from patients with BAV (n = 21) or tricuspid aortic valve (n = 12). Tissue samples were harvested from three circumferential regions corresponding to locations above the right, left, and noncoronary sinuses. Adventitial-stripped specimens containing media and intima only were analyzed for NO synthase 3 gene expression and total eNOS protein. Indicators of eNOS activity (pSer1177-eNOS) and NO bioavailability (phosphorylation of vasodilator-stimulated phosphoprotein at Ser239) were also measured. RESULTS: NO synthase 3 and eNOS protein were elevated in the right aortic region of BAV specimens compared with tricuspid aortic valve specimens. Activation of eNOS, as indicated by pSer1177-eNOS, was higher in BAV specimens across all regions. Despite increases in eNOS and pSer1177-eNOS, BAV specimens displayed no change in pSer239-vasodilator-stimulated phosphoprotein compared with tricuspid aortic valve specimens. CONCLUSIONS: BAV is associated with regional disruptions in the eNOS pathway, most markedly in the right aortic region. The discrepancy between increased eNOS activity and the absence of increased NO bioavailability in this region provides insight into physiologic mechanisms potentially underlying the asymmetric dilatation pattern observed in BAV

Background Bicuspid aortic valve (BAV) is associated with asymmetric dilatation of the proximal ascending aorta. We previously demonstrated increased susceptibility of smooth muscle cells to oxidative stress in the BAV-aneurysmal aorta and hypothesized that antioxidant expression is regionally defined and influenced by the BAV morphotype. Methods BAV valve morphology was defined according to number of raphes: type 0 (0 raphes), type 1 (1 raphe), or type 2 (2 raphes) and by the raphe location among the left (L), right (R) or non (N) coronary cusps. Ascending aortic specimens were partitioned into three regions corresponding to the sinuses of Valsalva, denoted R, N (greater curve), and L (lesser curve). Transcripts 1, 2, and 3 from the gene expressing superoxide dismutase ( Sod ) were quantified in all three regions. Results were compared with aneurysmal and nonaneurysmal aortic specimens from patients with a tricuspid aortic valve. Results Region-specific Sod1 upregulation and Sod2 downregulation were dependent on the BAV morphotype. Sod3 was uniformly downregulated in all regions in a morphotype-independent manner. Sod1 upregulation was noted in the R region of the nonaneurysmal type 1 L/R morphotype. Aortic valve regurgitation, but not stenosis, affected the expression of Sod isoforms in specimens of degenerative aneurysms. Conclusions Region-specific transcription profiles of Sod on the basis of BAV morphotype deepen our understanding of its associated aortopathy and provide biological insight on the asymmetric dilatation pattern. This work indicates regional differences exist in the oxidative stress biology of the proximal aortic wall, and this may lead to newer diagnostic techniques to adjudicate aortic catastrophe risk.

Introduction: Bicuspid aortic valve (BAV) is associated with an aortopathy that manifests as a dilatation of the proximal ascending aorta. The mechanisms of aneurysm formation in BAV patients are not completely understood. Our group established that medial smooth muscle cells (SMC) from aneurysmal BAV patients exhibit a weakened oxidative stress defense. Hypothesis: The weakened oxidative stress defense of medial SMCs in BAV patients leads to elevated oxidative stress in the proximal ascending aorta and decreases SMC viability. Methods: Aortic specimens were harvested from patients undergoing aortic valve and/or ascending aortic replacement due to aneurysm, or heart transplant, with IRB approval and informed patient consent. Levels of the superoxide anion-specific oxidation product 2-OH-E + was detected via HPLC in hydroethidine-exposed fresh human aortic specimens. Lipid peroxidation was quantified by ELISA-based detection of 8-iso-prostaglandin F2α. Superoxide anion (O 2 •- ) production and cell viability in primary SMC were detected using the cytochrome c reduction assay, and the MTT assay respectively. Results: Levels of O 2 •- and lipid peroxidation were elevated in BAV specimens when compared with tricuspid aortic valve (TAV) specimens (8.7 ± 0.90 vs 6.1 ± 0.90 pmol/mg, and 610±32 vs 458±45 pg/mg, Fig A and B respectively). Elevated O 2 •- production was detected in primary SMC in vitro when compared with SMC from TAV patients (13.2 ± 2.9 vs 6.7 ± 1.7 pmol/min/mg, Fig C). Addition of peroxide to primary SMC resulted in intracellular O 2 •- -mediated cell death (Fig D). Conclusions: Our results indicate that accumulation of O 2 •- in SMC is associated with increased oxidative stress and with increased cell death in the setting of BAV. Ongoing studies are focused on defining mechanisms of O 2 •- generation and the impact on aortic wall biomechanics. Importantly, this could help to develop novel interventions for BAV patients who are at risk for aortic catastrophe.

The suppression of transiently evoked otoacoustic emissions by contralateral sound stimulation is thought to occur as a result of the action of the efferent pathway from the superior olivary complex to the cochlea via the medial olivo-cochlear neurons. The purpose of this study was to determine the time taken for this pathway to activate the suppressive mechanism in response to contralateral sound in normal human subjects. The time for onset of suppression was found to be between 7 and 20 ms.

We have developed a method which can measure the menstrual prostaglandin (PG) activity in a single tampon specimen by bioassays. This method makes it possible to monitor the menstrual PG activity continuously during menstruation. Using this technique, we determined the menstrual PG patterns of two normal non-dysmenorrheic subjects, one subject on oral contraceptives (OC) and one subject with moderate to severe dysmenorrhea. Two to four cycles were studied per subject. We observed three mentrual patterns among the four subjects studied. Compared to the two normal controls, the subject on OC had a significantly lower menstrual fluid total and menstrual PG activity. The mean values +/- S.E. per menstrual period were 33.4 g +/- 1.5 vs 21.5 g +/- 2.0 and 28.6 microgram (PGF2alpha equivalent) +/- 1.5 vs 11.3 microgram +/- 4.2 respectively (control vs OC). The dysmenorrheic subject had a menstrual fluid total of 37.0 g +/- 1.9 similar to the two normal controls. Her menstrual PG activity (49.8 microgram +/- 7.7), however, was nearly two times higher than the normal controls. In one cycle studied, the dysmenorrheic subject was treated with a PG synthetase inhibitor, ibuprofen (Motrin). Remarkable relief was achieved. The alleviation of symptoms was accompanied by a concomitant marked reduction in the menstrual PG activity.

Extracellular matrix (ECM)-derived bioscaffolds have been shown to elicit tissue repair through retention of bioactive signals. Given that the adventitia of large blood vessels is a richly vascularized microenvironment, we hypothesized that perivascular ECM contains bioactive signals that influence cells of blood vessel lineages. ECM bioscaffolds were derived from decellularized human and porcine aortic adventitia (hAdv and pAdv, respectively) and then shown have minimal DNA content and retain elastin and collagen proteins. Hydrogel formulations of hAdv and pAdv ECM bioscaffolds exhibited gelation kinetics similar to ECM hydrogels derived from porcine small intestinal submucosa (pSIS). hAdv and pAdv ECM hydrogels displayed thinner, less undulated, and fibrous microarchitecture reminiscent of native adventitia, with slight differences in ultrastructure visible in comparison to pSIS ECM hydrogels. Pepsin-digested pAdv and pSIS ECM bioscaffolds increased proliferation of human adventitia-derived endothelial cells and this effect was mediated in part by basic fibroblast growth factor (FGF2). Human endothelial cells cultured on Matrigel substrates formed more numerous and longer tube-like structures when supplemented with pAdv ECM bioscaffolds, and FGF2 mediated this matrix signaling. ECM bioscaffolds derived from pAdv promoted FGF2-dependent in vivo angiogenesis in the chick chorioallantoic membrane model. Using an angiogenesis-focused protein array, we detected 55 angiogenesis-related proteins, including FGF2 in hAdv, pAdv and pSIS ECMs. Interestingly, 19 of these factors were less abundant in ECMs bioscaffolds derived from aneurysmal specimens of human aorta when compared with non-aneurysmal (normal) specimens. This study reveals that Adv ECM hydrogels recapitulate matrix fiber microarchitecture of native adventitia, and retain angiogenesis-related actors and bioactive properties such as FGF2 signaling capable of influencing processes important for angiogenesis. This work supports the use of Adv ECM bioscaffolds for both discovery biology and potential translation towards microvascular regeneration in clinical applications.