Off target toxicities and links with physicochemical properties of medicinal products, including antibiotics, oligonucleotides, lipid nanoparticles (with cationic and/or anionic charges). Data review suggests an emerging pattern.

Toxicology is an essential part of any drug development plan. Circumnavigating the risk of failure because of a toxicity issue can be a challenge, and failure in late development is extremely costly. To identify potential risks, it requires more than just understanding the biological target. The toxicologist needs to consider a compound's structure, it's physicochemical properties (including the impact of the overall formulation), as well as the biological target (e.g., receptor interactions). Understanding the impact of the physicochemical properties can be used to predict potential toxicities in advance by incorporating key endpoints in early screening strategies and/or used to compare toxicity profiles across lead candidates. This review discussed the risks of off-target and/or non-specific toxicities that may be associated with the physicochemical properties of compounds, especially those carrying dominant positive or negative charges, including amphiphilic small molecules, peptides, oligonucleotides and lipids/liposomes/lipid nanoparticles. The latter of which are being seen more and more in drug development, including the recent Covid pandemic, where mRNA and lipid nanoparticle technology is playing more of a role in vaccine development. The translation between non-clinical and clinical data is also considered, questioning how a physicochemical driven toxicity may be more universal across species, which means that such toxicity may be reassuringly translatable between species and as such, this information may also be considered as a support to the 3 R's, particularly in the early screening stages of a drug development plan.

Preclinical proof of concept for VivoVec, a lentiviral-based platform for in vivo CAR T-cell engineering.

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformational outcomes in the treatment of B-cell malignancies, but their widespread use is hindered by technical and logistical challenges associated with ex vivo cell manufacturing. To overcome these challenges, we developed VivoVec, a lentiviral vector-based platform for in vivo engineering of T cells. UB-VV100, a VivoVec clinical candidate for the treatment of B-cell malignancies, displays an anti-CD3 single-chain variable fragment (scFv) on the surface and delivers a genetic payload that encodes a second-generation CD19-targeted CAR along with a rapamycin-activated cytokine receptor (RACR) system designed to overcome the need for lymphodepleting chemotherapy in supporting successful CAR T-cell expansion and persistence. In the presence of exogenous rapamycin, non-transduced immune cells are suppressed, while the RACR system in transduced cells converts rapamycin binding to an interleukin (IL)-2/IL-15 signal to promote proliferation. METHODS: UB-VV100 was administered to peripheral blood mononuclear cells (PBMCs) from healthy donors and from patients with B-cell malignancy without additional stimulation. Cultures were assessed for CAR T-cell transduction and function. Biodistribution was evaluated in CD34-humanized mice and in canines. In vivo efficacy was evaluated against normal B cells in CD34-humanized mice and against systemic tumor xenografts in PBMC-humanized mice. RESULTS: In vitro, administration of UB-VV100 resulted in dose-dependent and anti-CD3 scFv-dependent T-cell activation and CAR T-cell transduction. The resulting CAR T cells exhibited selective expansion in rapamycin and antigen-dependent activity against malignant B-cell targets. In humanized mouse and canine studies, UB-VV100 demonstrated a favorable biodistribution profile, with transduction events limited to the immune compartment after intranodal or intraperitoneal administration. Administration of UB-VV100 to humanized mice engrafted with B-cell tumors resulted in CAR T-cell transduction, expansion, and elimination of systemic malignancy. CONCLUSIONS: These findings demonstrate that UB-VV100 generates functional CAR T cells in vivo, which could expand patient access to CAR T technology in both hematological and solid tumors without the need for ex vivo cell manufacturing.

Exploring the psychosocial needs of persons with lower extremity amputation and feasibility of internet cognitive behavioural therapy: a qualitative study.

PURPOSE: Following major lower extremity amputation (LEA), patients experience significant emotional distress and are at risk for anxiety and depression. There is a lack of mental health supports for this population, and internet-based cognitive behavioural therapy (iCBT) may be a useful resource to meet this need. The purpose of this study was to use a qualitative approach to explore the mental health needs of LEA patients and to gauge their attitudes of the use of iCBT to help them cope with their amputation. METHODS: Semi-structured qualitative interviews were conducted with inpatients and outpatients with LEA recruited from a major urban rehabilitation hospital. Data were analysed using inductive codebook thematic analysis (TA). RESULTS: Ten interviews were completed with individuals with LEA. The main themes identified were: (1) Fixating on the past; (2) Worry about the future; (3) Unmet mental health needs; (4) Barriers to Mental Health Support; (5) Importance of peer support; and (6) Tailoring iCBT. CONCLUSIONS: Our findings highlight that patients with LEA are open to learning more about iCBT to meet their mental health needs. Key iCBT implementation considerations include taking into account issues of stigma associated with mental health, timing of delivery, levels of digital literacy, online security, and interactive content.IMPLICATIONS FOR REHABILITATIONFollowing lower extremity amputation (LEA), people experience significant emotional distress and are at risk for the development of anxiety and/or depression.Patients with LEA are receptive to an online mental health resource (i.e., internet-based cognitive behavioural therapy [iCBT]) but it needs to be tailored to meet the various mental health needs and digital literacy of the LEA population.The use of an implementation science approach can help identify factors related to the development and potential uptake of an iCBT for patients with LEA.

A qualitative study on stakeholder perceptions of digital prosthetic socket fabrication for transtibial amputations.

BACKGROUND: Digital residual limb shape capture (three-dimensional [3D] scanning), computer-assisted design (CAD), and computer-assisted manufacturing with 3D printing technology show promise for a completely digital process of fabricating prosthetic sockets for patients with limb loss. The effectiveness and quality of digitally designed 3D-printed lower extremity prosthetic sockets is understudied, and there is lack of data on the patient and prosthetist experiences with this digital workflow. OBJECTIVE: To obtain stakeholder feedback on the feasibility and acceptability of using a completely digital prosthetic fabrication process consisting of 3D scanning, CAD, and 3D printing in a rehabilitation setting for adults with transtibial limb amputations. STUDY DESIGN: Qualitative design. METHODS: Study participants with a transtibial-level amputation were fit with a prosthetic socket fabricated using digital shape capture with a 3D scanner, CAD, and 3D printing in addition to a traditionally handcasted manually fabricated socket. Participants tried on and evaluated both sockets. Semistructured interviews took place after the fitting appointments. A focus group was conducted with prosthetists to obtain their feedback. Audio data were transcribed verbatim, and an inductive content analysis was undertaken. RESULTS: Eleven patient participants and 3 prosthetists identified 4 main themes: 1) openness and enthusiasm for digital prosthetic fabrication; 2) relative advantages of digital fabrication vs. traditional socket fabrication; 3) readiness of the technology used for adoption in practice; and 4) digital prosthetic workflow and 3D printing implementation considerations. CONCLUSIONS: Patients and prosthetists were enthusiastic about digital prosthetic socket fabrication and saw potential advantages over traditional methods. Both patients and prosthetists had concerns about the durability, safety, and aesthetics of the 3D printed sockets in this study. Further studies are needed to optimize digital prosthetic fabrication with 3D printing in prosthetic practice.

Implementing patient navigator programmes within a hospital setting in Toronto, Canada: A qualitative interview study.

OBJECTIVES: This study sought to identify the organisation and system level barriers and facilitators influencing the implementation of patient navigator programmes in one acute care hospital system in Toronto, Canada. METHODS: A qualitative descriptive approach informed by the Consolidated Framework for Implementation Research. Data were collected using in-depth interviews and analysed thematically. RESULTS: Thirty-eight individuals participated in interviews (17 community, 21 acute care hospital), including 24 frontline clinicians and 14 programme directors, health care leaders and managers. Implementation of patient navigator programmes was dependent on: (1) a clear consensus on the unique need for patient navigators; (2) champions to promote patient navigation; (3) programme ownership and accountability; (4) external system and organisational landscape and (5) implementation climate. Appropriate mechanisms of communication were found to have impacted each factor as a barrier or facilitator to programme implementation. CONCLUSION: Strategies for implementing patient navigator programmes into hospital clinical practice should include incorporating evidence to support the programme, considering mechanisms to enable collaborative communication, and the integration of frameworks to facilitate programme integration into the current practices within the organisation.

"We could be good partners if we work together": the perspectives of health and social service providers on the barriers to forming collaborative partnerships with social housing providers for older adults.

BACKGROUND: Many older adults are aging-at-home in social housing. However, the lack of integration between housing and health services makes it difficult for older tenants to access needed supports. We examined barriers and facilitators health and social service providers face providing on-site services to older tenants. METHODS: We conducted semi-structured qualitative interviews and focus groups with health and social service professionals (n = 58) in Toronto, Canada who provide community programs in support of older tenants who live in non-profit, rent-geared-to-income social housing. Interviews examined the barriers they faced in providing on-site services to older tenants. FINDINGS: Service providers strongly believed that collaboration with on-site housing staff led to better health and housing outcomes for older tenants. Despite the recognized benefits of partnering with housing staff, service providers felt that their ability to work effectively in the building was dependent on the staff (particularly the superintendent) assigned to that building. They also identified other barriers that made it difficult to work collaboratively with the housing provider, including staffing challenges such as high staff turnover and confusion about staff roles, a lack of understanding among housing staff about the link between housing and health, challenges sharing confidential information across sectors, and complex and inefficient partnership processes. CONCLUSION: Older adult tenants are increasingly vulnerable and in need of supports but the housing provider has a long history of ineffective partnerships with service providers driven by complex and inefficient staffing models, and an organizational culture that questions the role of and need for partnerships. Findings highlight the need for more effective integration of housing and health services. Simplified processes for establishing partnerships with service agencies and more opportunities for communication and collaboration with housing staff would ensure that services are reaching the most vulnerable tenants.

Perceptions of Risk: Perspectives on Crime and Safety in Public Housing for Older Adults.

BACKGROUND AND OBJECTIVES: An increasing number of older adults are aging in place in public housing. Public housing is perceived to have higher rates of crime that have detrimental impacts on health and well-being. We used a qualitative approach to understand the experiences of safety and unsafety for older adults in public housing. RESEARCH DESIGN AND METHODS: Participants included older adult tenants (n = 58) as well as service providers (n = 58) who offer supports directly in the buildings. Semistructured qualitative interviews and focus groups were used to explore (a) what makes the buildings feel unsafe, (b) how safety concerns affect access to support services, and (c) strategies used to promote safety. RESULTS: Participants acknowledged the importance of safety for creating a home-like environment; however, many described feeling unsafe at home or work. Participants described extreme examples of antisocial behaviors that were pervasive and viewed as commonplace. Lack of building security was a key issue, which was compounded by a perceived lack of accountability. While service providers were willing to accept a certain level of risk, many acknowledged that unsafe situations forced them to withdraw in-home services or stop community programs, further contributing to feelings of unsafety. In the absence of effective formal security, participants described several measures taken to mitigate risk. DISCUSSION AND IMPLICATIONS: Our findings point to the need for enhanced physical and environmental safety infrastructure, improved building management, increased on-site security, as well as other proactive measures to reduce risk by creating a greater sense of connection and community within the buildings.

Assessing readiness to implement patient navigator programs in Toronto, Canada.

OBJECTIVE: To explore factors influencing the implementation of patient navigator programs within a hospital for seniors with complex care needs. METHODS: A qualitative descriptive design using in-depth interviews was conducted. Participant interviews were conducted in Toronto, Ontario between September 2020 and February 2021. Data were analysed using thematic analysis. RESULTS: Thirty-five semi-structured interviews were conducted with 38 participants from a large urban hospital (n = 21) and community healthcare organizations (n = 17), including organizational leaders, and acute care and rehabilitation providers. Follow-up interviews occurred with 16 participants (7 from the community and nine from the hospital). This study identified five key factors influencing organizational readiness for successful implementation of a patient navigator program for seniors with complex conditions, which included: (a) vision from senior leadership, (b) technological infrastructure, (c) existing hospital-community partnerships, (d) well-established process for referrals, and (e) staff capacity. The overarching theme of communication was also identified. CONCLUSIONS: The findings of this study provide a better understanding of hospital and community professionals' needs and challenges when implementing patient navigator programs for seniors with complex care conditions. There are a number of factors that influence an organization's readiness for program uptake and delivery, with the need for clear communication being paramount. Further research to test the effects of readiness on successful implementation outcomes is warranted.

Production of copper-64 using a hospital cyclotron: targetry, purification and quality analysis.

OBJECTIVES: To construct and evaluate a 64Cu production system that minimises the amount of costly 64Ni, radionuclidic impurities and nonradioactive metal contamination and maximises radiochemical and radionuclidic purity and molar activity; and to report analytical and quality control methods that can be used within typical PET radiochemistry production facilities to measure metal ion concentrations and radiometal molar activities. METHODS: Low volume was ensured by dissolving the irradiated nickel in a low volume of hydrochloric acid (<1 mL) using the concave gold target backing as a reaction vessel in a custom-built target holder. Removal of contaminating 55Co and nonradioactive trace metals was ensured by adding an intermediate hydrochloric acid concentration step during the conventional ion-exchange elution process. The radionuclidic purity of the product was determined by half-life measurements, gamma spectroscopy and ion radiochromatography. Trace metal contamination and molar activity were determined by ion chromatography. RESULTS AND CONCLUSIONS: On a small scale, suitable for preclinical research, the process produced typically 3.2 GBq 64Cu in 2 mL solution from 9.4 ± 2.1 mg nickel-64 electroplated onto a gold target backing. The product had high molar activity (121.5 GBq/µmol), was free of trace metal contamination detectable by ion chromatography and has been used for many preclinical and clinical PET imaging applications.

Optimisation of CT protocols in PET-CT across different scanner models using different automatic exposure control methods and iterative reconstruction algorithms.

BACKGROUND: A significant proportion of the radiation dose from a PET-CT examination is dependent on the CT protocol, which should be optimised for clinical purposes. Matching protocols on different scanners within an imaging centre is important for the consistency of image quality and dose. This paper describes our experience translating low-dose CT protocols between scanner models utilising different automatic exposure control (AEC) methods and reconstruction algorithms. METHODS: The scanners investigated were a newly installed Siemens Biograph mCT PET with 64-slice SOMATOM Definition AS CT using sinogram affirmed iterative reconstruction (SAFIRE) and two GE Discovery 710 PET scanners with 128-slice Optima 660 CT using adaptive statistical reconstruction (ASiR). Following exploratory phantom work, 33 adult patients of various sizes were scanned using the Siemens scanner and matched to patients scanned using our established GE protocol to give 33 patient pairs. A comparison of volumetric CT dose index (CTDIvol) and image noise within these patient pairs informed optimisation, specifically for obese patients. Another matched patient study containing 27 patient pairs was used to confirm protocol matching. Size-specific dose estimates (SSDEs) were calculated for patients in the second cohort. With the acquisition protocol for the Siemens scanner determined, clinicians visually graded the images to identify optimal reconstruction parameters. RESULTS: In the first matched patient study, the mean percentage difference in CTDIvol for Siemens compared to GE was - 10.7% (range - 41.7 to 50.1%), and the mean percentage difference in noise measured in the patients' liver was 7.6% (range - 31.0 to 76.8%). In the second matched patient study, the mean percentage difference in CTDIvol for Siemens compared to GE was - 20.5% (range - 43.1 to 1.9%), and the mean percentage difference in noise was 19.8% (range - 27.0 to 146.8%). For these patients, the mean SSDEs for patients scanned on the Siemens and GE scanners were 3.27 (range 2.83 to 4.22) mGy and 4.09 (range 2.81 to 4.82) mGy, respectively. The analysis of the visual grading study indicated no preference for any of the SAFIRE strengths. CONCLUSIONS: Given the different implementations of acquisition parameters and reconstruction algorithms between vendors, careful consideration is required to ensure optimisation and standardisation of protocols.

Health care access and use among male and female Canadian Armed Forces veterans.

BACKGROUND: Veterans are more likely than other Canadians to have chronic health conditions, making access to health care an important issue. However, little research has addressed health care access and use among veterans. This paper examines access and use among veterans compared with other Canadians. DATA AND METHODS: Health care access and use indicators were examined for Regular Force veterans using the 2016 Life After Service Survey. Information for male and female veterans was compared with information on the Canadian general population from the 2015 and 2016 Canadian Community Health Survey, using age-adjusted rates and 95% confidence intervals. RESULTS: More than 80% of male and female veterans reported having a regular medical doctor in the 12 months before the survey. The majority of veterans (71% of males and 81% of females) had consulted a family doctor, while a minority had been hospitalized (8% of males and females). These rates were similar to those in the Canadian general population. However, veteran consultation rates for mental health care and with audiologists, speech therapists or occupational therapists among both sexes were double to triple those of the Canadian general population. Among veterans, males reported lower rates of unmet needs compared with females. DISCUSSION: Veterans had similar rates of access to a regular medical doctor and higher rates of use compared with other Canadians. However, these may be comparatively low, given previous findings on higher rates of disability and some chronic conditions among veterans. Noted differences between males and females highlight the importance of research and services that account for sex and gender. The extent to which health care needs explain health care use and barriers to care requires further research.

Simultaneous 13N-Ammonia and gadolinium first-pass myocardial perfusion with quantitative hybrid PET-MR imaging: a phantom and clinical feasibility study.

BACKGROUND: Positron emission tomography (PET) is the non-invasive reference standard for myocardial blood flow (MBF) quantification. Hybrid PET-MR allows simultaneous PET and cardiac magnetic resonance (CMR) acquisition under identical experimental and physiological conditions. This study aimed to determine feasibility of simultaneous 13N-Ammonia PET and dynamic contrast-enhanced CMR MBF quantification in phantoms and healthy volunteers. METHODS: Images were acquired using a 3T hybrid PET-MR scanner. Phantom study: MBF was simulated at different physiological perfusion rates and a protocol for simultaneous PET-MR perfusion imaging was developed. Volunteer study: five healthy volunteers underwent adenosine stress. 13N-Ammonia and gadolinium were administered simultaneously. PET list mode data was reconstructed using ordered subset expectation maximisation. CMR MBF was quantified using Fermi function-constrained deconvolution of arterial input function and myocardial signal. PET MBF was obtained using a one-tissue compartment model and image-derived input function. RESULTS: Phantom study: PET and CMR MBF measurements demonstrated high repeatability with intraclass coefficients 0.98 and 0.99, respectively. There was high correlation between PET and CMR MBF (r = 0.98, p < 0.001) and good agreement (bias - 0.85 mL/g/min; 95% limits of agreement 0.29 to - 1.98). Volunteer study: Mean global stress MBF for CMR and PET were 2.58 ± 0.11 and 2.60 ± 0.47 mL/g/min respectively. On a per territory basis, there was moderate correlation (r = 0.63, p = 0.03) and agreement (bias - 0.34 mL/g/min; 95% limits of agreement 0.49 to - 1.18). CONCLUSION: Simultaneous MBF quantification using hybrid PET-MR imaging is feasible with high test repeatability and good to moderate agreement between PET and CMR. Future studies in coronary artery disease patients may allow cross-validation of techniques.

Role of cell-matrix interactions on VIC phenotype and tissue deposition in 3D PEG hydrogels.

Valvular interstitial cells (VICs) respond to 3D matrix interactions in a complex manner, but understanding these effects on VIC function better is important for applications ranging from valve tissue engineering to studying valve disease. Here, we encapsulated VICs in poly(ethylene glycol) (PEG) hydrogels modified with three different adhesive ligands, derived from fibronectin (RGDS), elastin (VGVAPG) and collagen-1 (P15). By day 14, VICs became significantly more elongated in RGDS-containing gels compared to VGVAPG or P15. This difference in cell morphology appeared to correlate with global matrix metalloproteinase (MMP) activity, as VICs encapsulated in RGDS-functionalized hydrogels secreted higher levels of active MMP at day 2. VIC activation to a myofibroblast phenotype was also characterized by staining for α-smooth muscle actin (αSMA) at day 14. The percentage of αSMA+ VICs in the VGVAPG gels was the highest (56%) compared to RGDS (33%) or P15 (38%) gels. Matrix deposition and composition were also characterized at days 14 and 42 and found to depend on the initial hydrogel composition. All gel formulations had similar levels of collagen, elastin and chondroitin sulphate deposited as the porcine aortic valve. However, the composition of collagen deposited by VICs in VGVAPG-functionalized gels had a significantly higher collagen-X:collagen-1 ratio, which is associated with stenotic valves. Taken together, these data suggest that peptide-functionalized PEG hydrogels are a useful system for culturing VICs three-dimensionally and, with the ability to systematically alter biochemical and biophysical properties, this platform may prove useful in manipulating VIC function for valve regeneration. Copyright © 2013 John Wiley & Sons, Ltd.

Safety of vaccine adjuvants: focus on autoimmunity.

Questions have been recently raised regarding the safety of vaccine adjuvants, particularly in relation to autoimmunity or autoimmune disease(s)/disorder(s) (AID). The International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) formed a scientific committee and convened a 2-day workshop, consisting of technical experts from around the world representing academia, government regulatory agencies, and industry, to investigate and openly discuss the issues around adjuvant safety in vaccines. The types of adjuvants considered included oil-in-water emulsions and toll-like receptor (TLR) agonists. The state of science around the use of animal models and biomarkers for the evaluation and prediction of AID were also discussed. Following extensive literature reviews by the HESI committee, and presentations by experts at the workshop, several key points were identified, including the value of animal models used to study autoimmunity and AID toward studying novel vaccine adjuvants; whether there is scientific evidence indicating an intrinsic risk of autoimmunity and AID with adjuvants, or a higher risk resulting from the mechanism of action; and if there is compelling clinical data linking adjuvants and AID. The tripartite group of experts concluded that there is no compelling evidence supporting the association of vaccine adjuvants with autoimmunity signals. Additionally, it is recommended that future research on the potential effects of vaccine adjuvants on AID should consider carefully the experimental design in animal models particularly if they are to be used in any risk assessment, as an improper design and model could result in misleading information. Finally, studies on the mechanistic aspects and potential biomarkers related to adjuvants and autoimmunity phenomena could be developed.

Aortic valve sclerosis in mice deficient in endothelial nitric oxide synthase.

Risk factors for fibrocalcific aortic valve disease (FCAVD) are associated with systemic decreases in bioavailability of endothelium-derived nitric oxide (EDNO). In patients with bicuspid aortic valve (BAV), vascular expression of endothelial nitric oxide synthase (eNOS) is decreased, and eNOS(-/-) mice have increased prevalence of BAV. The goal of this study was to test the hypotheses that EDNO attenuates profibrotic actions of valve interstitial cells (VICs) in vitro and that EDNO deficiency accelerates development of FCAVD in vivo. As a result of the study, coculture of VICs with aortic valve endothelial cells (vlvECs) significantly decreased VIC activation, a critical early phase of FCAVD. Inhibition of VIC activation by vlvECs was attenuated by N(G)-nitro-l-arginine methyl ester or indomethacin. Coculture with vlvECs attenuated VIC expression of matrix metalloproteinase-9, which depended on stiffness of the culture matrix. Coculture with vlvECs preferentially inhibited collagen-3, compared with collagen-1, gene expression. BAV occurred in 30% of eNOS(-/-) mice. At age 6 mo, collagen was increased in both bicuspid and trileaflet eNOS(-/-) aortic valves, compared with wild-type valves. At 18 mo, total collagen was similar in eNOS(-/-) and wild-type mice, but collagen-3 was preferentially increased in eNOS(-/-) mice. Calcification and apoptosis were significantly increased in BAV of eNOS(-/-) mice at ages 6 and 18 mo. Remarkably, these histological changes were not accompanied by physiologically significant valve stenosis or regurgitation. In conclusion, coculture with vlvECs inhibits specific profibrotic VIC processes. In vivo, eNOS deficiency produces fibrosis in both trileaflet and BAVs but produces calcification only in BAVs.

Clickable, photodegradable hydrogels to dynamically modulate valvular interstitial cell phenotype.

Biophysical cues are widely recognized to influence cell phenotype. While this evidence was established using static substrates, there is growing interest in creating stimulus-responsive biomaterials that better recapitulate the dynamic extracellular matrix. Here, a clickable, photodegradable hydrogel substrate that allows the user to precisely control substrate elasticity and topography in situ is presented. The hydrogels are synthesized by reacting an 8-arm poly(ethylene glycol) alkyne with an azide-functionalized photodegradable crosslinker. The utility of this platform by exploiting its photoresponsive properties to modulate the phenotype of porcine aortic valvular interstitial cells (VICs) is demonstrated. First, VIC phenotype is monitored, in response to initial substratum modulus and static topographic cues. Higher modulus (E ≈ 15 kPa) substrates induce higher levels of activation (≈70% myofibroblasts) versus soft (E ≈ 3 kPa) substrates (≈20% myofibroblasts). Microtopographies that induce VIC alignment and elongation on low modulus substrates also stimulate activation. Finally, VIC phenotype is monitored in response to sequential in situ manipulations. The results illustrate that VIC activation on stiff surfaces (≈70% myofibroblasts) can be partially reversed by reducing surface modulus (≈30% myofibroblats) and subsequently re-activated by anisotropic topographies (≈60% myofibroblasts). Such dynamic substrates afford unique opportunities to decipher the complex role of matrix cues on the plasticity of VIC activation.

The role of valvular endothelial cell paracrine signaling and matrix elasticity on valvular interstitial cell activation.

The effects of valvular endothelial cell (VlvEC) paracrine signaling on VIC phenotype and nodule formation were tested using a co-culture platform with physiologically relevant matrix elasticities and diffusion distance. 100 µm thin poly(ethylene glycol) (PEG) hydrogels of 3-27 kPa Young's moduli were fabricated in transwell inserts. VICs were cultured on the gels, as VIC phenotype is known to change significantly within this range, while VlvECs lined the underside of the membrane. Co-culture with VlvECs significantly reduced VIC activation to the myofibroblast phenotype on all gels with the largest percent decrease on the 3 kPa gels (~70%), while stiffer gels resulted in approximately 20-30% decrease. Additionally, VlvECs significantly reduced αSMA protein expression (~2 fold lower) on both 3 and 27 kPa gels, as well as the number (~2 fold lower) of nodules formed on the 27 kPa gels. Effects of VlvECs were prevented when nitric oxide (NO) release was inhibited with l-NAME, suggesting that VlvEC produced NO inhibits VIC activation. Withdrawal of l-NAME after 3, 5, and 7 days with restoration of VlvEC NO production for 2 additional days led to a partial reversal of VIC activation (~25% decrease). A potential mechanism by which VlvEC produced NO reduced VIC activation was studied by inhibiting initial and mid-stage cGMP pathway molecules. Inhibition of soluble guanylyl cyclase (sGC) with ODQ or protein kinase G (PKG) with RBrcGMP or stimulation of Rho kinase (ROCK) with LPA, abolished VlvEC effects on VIC activation. This work contributes substantially to the understanding of the valve endothelium's role in preventing VIC functions associated with aortic valve stenosis initiation and progression.

Hemodynamic and cellular response feedback in calcific aortic valve disease.

This review highlights aspects of calcific aortic valve disease that encompass the entire range of aortic valve disease progression from initial cellular changes to aortic valve sclerosis and stenosis, which can be initiated by changes in blood flow (hemodynamics) and pressure across the aortic valve. Appropriate hemodynamics is important for normal valve function and maintenance, but pathological blood velocities and pressure can have profound consequences at the macroscopic to microscopic scales. At the macroscopic scale, hemodynamic forces impart shear stresses on the surface of the valve leaflets and cause deformation of the leaflet tissue. As discussed in this review, these macroscale forces are transduced to the microscale, where they influence the functions of the valvular endothelial cells that line the leaflet surface and the valvular interstitial cells that populate the valve extracellular matrix. For example, pathological changes in blood flow-induced shear stress can cause dysfunction, impairing their homeostatic functions, and pathological stretching of valve tissue caused by elevated transvalvular pressure can activate valvular interstitial cells and latent paracrine signaling cytokines (eg, transforming growth factor-ß1) to promote maladaptive tissue remodeling. Collectively, these coordinated and complex interactions adversely impact bulk valve tissue properties, feeding back to further deteriorate valve function and propagate valve cell pathological responses. Here, we review the role of hemodynamic forces in calcific aortic valve disease initiation and progression, with focus on cellular responses and how they feed back to exacerbate aortic valve dysfunction.

Small peptide functionalized thiol-ene hydrogels as culture substrates for understanding valvular interstitial cell activation and de novo tissue deposition.

A thiol-ene polymerization platform was used to synthesize peptide functionalized poly(ethylene glycol) hydrogels, which were initially characterized and compared to theoretical predictions of Young's modulus via a theoretical crosslinking density equation presented herein. After thorough characterization, this material system's utility for answering specific biological hypotheses was demonstrated with the culture and observation of aortic valvular interstitial cells (VICs). Specifically, these materials were used to better understand the role of substrate elasticity and biochemical functionality on VIC α-smooth muscle (αSMA) expression and secretory properties (i.e. de novo extracellular matrix (ECM)). The Young's moduli of the hydrogels varied from 28kPa (activating, 90% myofibroblasts) to 4kPa (non-activating, 15% myofibroblast), and the biochemical functionality was tailored by incorporating three small adhesive peptide sequences, RGDS, VGVAPG and P15. To promote VIC adhesion, a basal [RGDS] of 0.8mM was used in all formulations, while the [VGVAPG] or [P15] were varied to be lower than, equal to or higher than 0.8mM. The substrates with 1.2mM VGVAPG and all gels with P15 led to significantly higher αSMA expression for both stiff and soft substrates, as compared to 0.8mM RGDS alone. Importantly, all gel conditions αSMA expression were significantly lower than tissue culture poly(styrene) (TCPS; ∼4- to 10-fold difference). The ECM produced decreased significantly as the total integrin-binding peptide concentration increased, but was significantly higher than that produced on TCPS. This easily tailored material system provides a useful culture platform to improve the fundamental understanding of VIC biology through isolating specific biological cues and observing VIC function.