Lysyl oxidase expression in cardiac fibroblasts is regulated by α2ß1 integrin interactions with the cellular microenvironment.

Lysyl oxidase (LOX) catalyzes crosslink formation between fibrillar collagens and elastins and an increase in LOX activity has been associated with cardiac fibrosis following myocardial infarction (MI). It has been previously reported that LOX expression is regulated by growth factors and cytokines including transforming growth factor (TGF-ß1); however, it is unclear how the biophysical and biochemical properties of the cellular microenvironment affect LOX expression. In this study, we isolated rat cardiac fibroblasts (CF) and infarct cardiac fibroblasts (ICF), from healthy and 1-week post-MI left ventricular tissue respectively, and cultured them under varied substrate conditions in vitro to assess their influence on LOX expression. Culture of ICF on collagen I-coated plates increased LOX expression versus uncoated plates with an additional increase observed with the presence of TGF-ß1. To further investigate the effect of integrin interactions with collagen I on LOX expression, we inhibited the α2ß1 integrin from binding to collagen I and found gene and protein expression of LOX to be downregulated. Together, this demonstrates that the interaction of α2ß1 integrin to collagen I in the cellular microenvironment can regulate expression of LOX. Further studies investigating additional integrin interactions may identify therapeutic targets for treating cardiac fibrosis.

An in vitro model for the assessment of stem cell fate following implantation within the infarct microenvironment identifies ISL-1 expression as the strongest predictor of c-Kit(+) cardiac progenitor cells' therapeutic potential.

Cell therapy has the potential to drastically improve clinical outcomes for the 1.45 million patients suffering from a myocardial infarction (MI) each year in the U.S. However, the limitations associated with this treatment - including poor engraftment, significant cell death and poor differentiation potential - have prevented its widespread application clinically. To optimize functional improvements provided by transplanted cells, there is a need to develop methods that increase cellular retention and viability, while supporting differentiation and promoting paracrine signaling. Current in vivo models are expensive, difficult to access and manipulate and are time consuming. We have developed an in vitro model of MI which allows for a straightforward, consistent and relatively accurate prediction of cell fate following injection in vivo. The model demonstrated how the infarct environment impairs cellular engraftment and differentiation, but identified an implantation strategy which enhanced cell fate in vitro. Multivariate linear regression identified variables within the model that regulated vascular differentiation potential including oxygen tension, stiffness and cytokine presence, while cardiac differentiation was more accurately predicted by Isl-1 expression in the original cell isolate than any other variable present within the model system. The model highlighted how the cells' sensitivity to the infarct variables varied from line to line, which emphasizes the importance of the model system for the prediction of cell fate on a patient specific basis. Further development of this model system could help predict the clinical efficacy of cardiac progenitor cell therapy at the patient level as well as identify the optimal strategy for cell delivery.

Moving the needle: Quality improvement strategies to achieve guideline-concordant care of obstetric patients with severe hypertension.

OBJECTIVES: To improve timely treatment and follow-up of birthing individuals with severe hypertension. STUDY DESIGN: A quality improvement (QI) initiative was implemented at an academic tertiary care center in the United States of America for individuals with obstetric hypertensive emergencies. Statistical process control charts were utilized to track process measures and interventions tested through plan-do-study-act cycles. Measures were disaggregated by race and ethnicity to identify and improve disparities. MAIN OUTCOME MEASURES: Treatment of hypertensive events within 60 min, receipt of blood pressure (BP) device at discharge and completed postpartum follow-up BP check within 7 days of discharge. RESULTS: All process measures showed statistically significant improvements. The primary process measure, timely treatment of hypertensive emergencies, improved from 29 % to 76 %. Receipt of BP device improved from 37 % to 91 % and follow-up BP checks from 58 % to 81 %. No racial or ethnic disparities were noted at baseline or after interventions. Readmission rates within 6 weeks of delivery increased from 2.3 % to 6.1 % for the cohort with no severe morbidity or mortality events after discharge. Strategies associated with improvement included project launch with establishment of the "why," telehealth, simulation, a video display of quality metrics on the birthing unit, promoting BP cuff access, and automated orders. CONCLUSIONS: This comprehensive QI initiative provides novel improvement strategies for the management of individuals with severe hypertensive disorders of pregnancy for the timely treatment of severe BP, attainment of home BP devices, and follow-up after discharge. Quality improvement methodology is practical and essential for achieving guideline-concordant care.

Mesenchymal stem cells ability to generate traction stress in response to substrate stiffness is modulated by the changing extracellular matrix composition of the heart during development.

In this study we present a novel method for studying cellular traction force generation and mechanotransduction in the context of cardiac development. Rat hearts from three distinct stage of development (fetal, neonatal and adult) were isolated, decellularized and characterized via mechanical testing and protein compositional analysis. Stiffness increased ~2-fold between fetal and neonatal time points but not between neonatal and adult. Composition of structural extracellular matrix (ECM) proteins was significantly different between all three developmental ages. ECM that was solubilized via pepsin digestion was cross-linked into polyacrylamide gels of varying stiffness and traction force microscopy was used to assess the ability of mesenchymal stem cells (MSCs) to generate traction stress against the substrates. The response to increasing stiffness was significantly different depending on the developmental age of the ECM. An investigation into early cardiac differentiation of MSCs demonstrated a dependence of the level of expression of early cardiac transcription factors on the composition of the complex ECM. In summary, this study found that complex ECM composition plays an important role in modulating a cell's ability to generate traction stress against a substrate, which is a significant component of mechanotransductive signaling.

Extracellular matrix and cyclic stretch alter fetal cardiomyocyte proliferation and maturation in a rodent model of heart hypoplasia.

Introduction: Birth defects, particularly those that affect development of the heart, are a leading cause of morbidity and mortality in infants and young children. Babies born with heart hypoplasia (heart hypoplasia) disorders often have a poor prognosis. It remains unclear whether cardiomyocytes from hypoplastic hearts retain the potential to recover growth, although this knowledge would be beneficial for developing therapies for heart hypoplasia disorders. The objective of this study was to determine the proliferation and maturation potential of cardiomyocytes from hypoplastic hearts and whether these behaviors are influenced by biochemical signaling from the extracellular matrix (ECM) and cyclic mechanical stretch. Method: Congenital diaphragmatic hernia (CDH)-associated heart hypoplasia was induced in rat fetuses by maternal exposure to nitrofen. Hearts were isolated from embryonic day 21 nitrofen-treated fetuses positive for CDH (CDH+) and from fetuses without nitrofen administration during gestation. Results and discussion: CDH+ hearts were smaller and had decreased myocardial proliferation, along with evidence of decreased maturity compared to healthy hearts. In culture, CDH+ cardiomyocytes remained immature and demonstrated increased proliferative capacity compared to their healthy counterparts. Culture on ECM derived from CDH+ hearts led to a significant reduction in proliferation for both CDH+ and healthy cardiomyocytes. Healthy cardiomyocytes were dosed with exogenous nitrofen to examine whether nitrofen may have an aberrant effect on the proliferative ability of cardiomyocyte, yet no significant change in proliferation was observed. When subjected to stretch, CDH+ cardiomyocytes underwent lengthening of sarcomeres while healthy cardiomyocyte sarcomeres were unaffected. Taken together, our results suggest that alterations to environmental cues such as ECM and stretch may be important factors in the pathological progression of heart hypoplasia.

Uncovering the roles of dihydropyrimidine dehydrogenase in fatty-acid induced steatosis using human cellular models.

Pyrimidine catabolism is implicated in hepatic steatosis. Dihydropyrimidine dehydrogenase (DPYD) is an enzyme responsible for uracil and thymine catabolism, and DPYD human genetic variability affects clinically observed toxicity following 5-Fluorouracil administration. In an in vitro model of fatty acid-induced steatosis, the pharmacologic inhibition of DPYD resulted in protection from lipid accumulation. Additionally, a gain-of-function mutation of DPYD, created through clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR-Cas9) engineering, led to an increased lipid burden, which was associated with altered mitochondrial functionality in a hepatocarcionma cell line. The studies presented herein describe a novel role for DPYD in hepatocyte metabolic regulation as a modulator of hepatic steatosis.

Incorporation of 2,3-diaminopropionic acid into linear cationic amphipathic peptides produces pH-sensitive vectors.

Nonviral vectors that harness the change in pH in endosomes, are increasingly being used to deliver cargoes, including nucleic acids, into mammalian cells. Here we present evidence that the pK(a) of the beta-NH(2) in 2,3-diaminopropionic acid (Dap) is sufficiently lowered, when Dap is incorporated into peptides, that its protonation state is sensitive to the pH changes that occur during endosomal acidification. The lowered pK(a) of around 6.3 is stabilized by the increased electron-withdrawing effect of the peptide bonds, by intermolecular hydrogen bonding and from contributions arising from the peptide conformation. These include mixed polar/apolar environments, Coulombic interactions and intermolecular hydrogen bonding. Changes in the charged state are therefore expected between pH 5 and 7, and large-scale conformational changes are observed in Dap-rich peptides, in contrast to analogues containing lysine or ornithine, when the pH is altered through this range. These physical properties confer a robust gene-delivery capability on designed cationic amphipathic peptides that incorporate Dap.

The stem cell/cancer stem cell marker ALDH1A3 regulates the expression of the survival factor tissue transglutaminase, in mesenchymal glioma stem cells.

Tissue transglutaminase (tTG), a dual-function enzyme with GTP-binding and acyltransferase activities, has been implicated in the survival and chemotherapy resistance of aggressive cancer cells and cancer stem cells, including glioma stem cells (GSCs). Using a model system comprising two distinct subtypes of GSCs referred to as proneural (PN) and mesenchymal (MES), we find that the phenotypically aggressive and radiation therapy-resistant MES GSCs exclusively express tTG relative to PN GSCs. As such, the self-renewal, proliferation, and survival of these cells was sensitive to treatment with tTG inhibitors, with a benefit being observed when combined with the standard of care for high grade gliomas (i.e. radiation or temozolomide). Efforts to understand the molecular drivers of tTG expression in MES GSCs revealed an unexpected link between tTG and a common marker for stem cells and cancer stem cells, Aldehyde dehydrogenase 1A3 (ALDH1A3). ALDH1A3, as well as other members of the ALDH1 subfamily, can function in cells as a retinaldehyde dehydrogenase to generate retinoic acid (RA) from retinal. We show that the enzymatic activity of ALDH1A3 and its product, RA, are necessary for the observed expression of tTG in MES GSCs. Additionally, the ectopic expression of ALDH1A3 in PN GSCs is sufficient to induce the expression of tTG in these cells, further demonstrating a causal link between ALDH1A3 and tTG. Together, these findings ascribe a novel function for ALDH1A3 in an aggressive GSC phenotype via the up-regulation of tTG, and suggest the potential for a similar role by ALDH1 family members across cancer types.

In vitro and in vivo analysis of visible light crosslinkable gelatin methacryloyl (GelMA) hydrogels.

Photocrosslinkable materials have been frequently used for constructing soft and biomimetic hydrogels for tissue engineering. Although ultraviolet (UV) light is commonly used for photocrosslinking such materials, its use has been associated with several biosafety concerns such as DNA damage, accelerated aging of tissues, and cancer. Here we report an injectable visible light crosslinked gelatin-based hydrogel for myocardium regeneration. Mechanical characterization revealed that the compressive moduli of the engineered hydrogels could be tuned in the range of 5-56 kPa by changing the concentrations of the initiator, co-initiator and co-monomer in the precursor formulation. In addition, the average pore sizes (26-103 µm) and swelling ratios (7-13%) were also shown to be tunable by varying the hydrogel formulation. In vitro studies showed that visible light crosslinked GelMA hydrogels supported the growth and function of primary cardiomyocytes (CMs). In addition, the engineered materials were shown to be biocompatible in vivo, and could be successfully delivered to the heart after myocardial infarction in an animal model to promote tissue healing. The developed visible light crosslinked hydrogel could be used for the repair of various soft tissues such as the myocardium and for the treatment of cardiovascular diseases with enhanced therapeutic functionality.

Optical metrics of the extracellular matrix predict compositional and mechanical changes after myocardial infarction.

Understanding the organization and mechanical function of the extracellular matrix (ECM) is critical for the development of therapeutic strategies that regulate wound healing following disease or injury. However, these relationships are challenging to elucidate during remodeling following myocardial infarction (MI) due to rapid changes in cellularity and an inability to characterize both ECM microstructure and function non-destructively. In this study, we overcome those challenges through whole organ decellularization and non-linear optical microscopy to directly relate the microstructure and mechanical properties of myocardial ECM. We non-destructively quantify collagen organization, content, and cross-linking within decellularized healthy and infarcted myocardium using second harmonic generation (SHG) and two photon excited autofluorescence. Tensile mechanical testing and compositional analysis reveal that the cumulative SHG intensity within each image volume and the average collagen autofluorescence are significantly correlated with collagen content and elastic modulus of the ECM, respectively. Compared to healthy ECM, infarcted tissues demonstrate a significant increase in collagen content and fiber alignment, and a decrease in cross-linking and elastic modulus. These findings indicate that cross-linking plays a key role in stiffness at the collagen fiber level following infarction, and highlight how this non-destructive approach to assessing remodeling can be used to understand ECM structure-function relationships.

Cardiac extracellular matrix-fibrin hybrid scaffolds with tunable properties for cardiovascular tissue engineering.

Solubilized cardiac extracellular matrix (ECM) is being developed as an injectable therapeutic that offers promise for promoting cardiac repair. However, the ECM alone forms a hydrogel that is very soft compared to the native myocardium. As both the stiffness and composition of the ECM are important in regulating cell behavior and can have complex synergistic effects, we sought to develop an ECM-based scaffold with tunable biochemical and mechanical properties. We used solubilized rat cardiac ECM from two developmental stages (neonatal, adult) combined with fibrin hydrogels that were cross-linked with transglutaminase. We show that ECM was retained within the gels and that the Young's modulus could be tuned to span the range of the developing and mature heart. C-kit+ cardiovascular progenitor cells from pediatric patients with congenital heart defects were seeded into the hybrid gels. Both the elastic modulus and composition of the scaffolds impacted the expression of endothelial and smooth muscle cell genes. Furthermore, we demonstrate that the hybrid gels are injectable, and thus have potential for minimally invasive therapies. ECM-fibrin hybrid scaffolds offer new opportunities for exploiting the effects of both composition and mechanical properties in directing cell behavior for tissue engineering.

Differences between perception and eye movements during complex motions.

During passive whole-body motion in the dark, the motion perceived by subjects may or may not be veridical. Either way, reflexive eye movements are typically compensatory for the perceived motion. However, studies are discovering that for certain motions, the perceived motion and eye movements are incompatible. The incompatibility has not been explained by basic differences in gain or time constants of decay. This paper uses three-dimensional modeling to investigate gondola centrifugation (with a tilting carriage) and off-vertical axis rotation. The first goal was to determine whether known differences between perceived motions and eye movements are true differences when all three-dimensional combinations of angular and linear components are considered. The second goal was to identify the likely areas of processing in which perceived motions match or differ from eye movements, whether in angular components, linear components and/or dynamics. The results were that perceived motions are more compatible with eye movements in three dimensions than the one-dimensional components indicate, and that they differ more in their linear than their angular components. In addition, while eye movements are consistent with linear filtering processes, perceived motion has dynamics that cannot be explained by basic differences in time constants, filtering, or standard GIF-resolution processes.