The renin-angiotensin system (RAS) is activated in aortic valve disease, yet little is understood about how it affects the acute functional response of valve interstitial cells (VICs). Herein, we ...developed a gelatin-based valve thin film (vTF) platform to investigate whether the contractile response of VICs can be regulated
via
RAS mediators and inhibitors. First, the impact of culture medium (quiescent, activated, and osteogenic medium) on VIC phenotype and function was assessed. Contractility of VICs was measured upon treatment with angiotensin I (Ang I), angiotensin II (Ang II), angiotensin-converting enzyme (ACE) inhibitor, and Angiotensin II type 1 receptor (AT
1
R) inhibitor. Anisotropic cell alignment on gelatin vTF was achieved independent of culture conditions. Cells cultured in activated and osteogenic conditions were found to be more elongated than in quiescent medium. Increased α-SMA expression was observed in activated medium and no RUNX2 expression were observed in cells. VIC contractile stress increased with increasing concentrations (from 10
−10
to 10
−6
M) of Ang I and Ang II. Moreover, cell contraction was significantly reduced in all ACE and AT
1
R inhibitor-treated groups. Together, these findings suggest that local RAS is active in VICs, and our vTF may provide a powerful platform for valve drug screening and development.
The blood-brain barrier (BBB) is a physiological regulator of transport of essential items from blood to brain for the maintenance of homeostasis of the central nervous system (CNS) within narrow ...limits. The BBB is also responsible for export of harmful or metabolic products from brain to blood to keep the CNS fluid microenvironment healthy. However, noxious insults to the brain caused by trauma, ischemia or environmental/chemical toxins alter the BBB function to small as well as large molecules e.g., proteins. When proteins enter the CNS fluid microenvironment, development of brain edema occurs due to altered osmotic balance between blood and brain. On the other hand, almost all neurodegenerative diseases and traumatic insults to the CNS and subsequent BBB dysfunction lead to edema formation and cell injury. To treat these brain disorders suitable drug therapy reaching their brain targets is needed. However, due to edema formation or only a focal disruption of the BBB e.g., around brain tumors, many drugs are unable to reach their CNS targets in sufficient quantity. This results in poor therapeutic outcome. Thus, new technology such as nanodelivery is needed for drugs to reach their CNS targets and be effective. In this review, use of nanowires as a possible novel tool to enhance drug delivery into the CNS in various disease models is discussed based on our investigations. These data show that nanowired delivery of drugs may have superior neuroprotective ability to treat several CNS diseases effectively indicating their role in future therapeutic strategies.
Calcific aortic valve disease (CAVD) pathophysiology is a complex, multistage process, usually diagnosed at advanced stages after significant anatomical and hemodynamic changes in the valve. Early ...detection of disease progression is thus pivotal in the development of prevention and mitigation strategies. In this study, we developed a diet-based, non-genetically modified mouse model for early CAVD progression, and explored the utility of two-photon excited fluorescence (TPEF) microscopy for early detection of CAVD progression. TPEF imaging provides label-free, non-invasive, quantitative metrics with the potential to correlate with multiple stages of CAVD pathophysiology including calcium deposition, collagen remodeling and osteogenic differentiation.
Twenty-week old C57BL/6J mice were fed either a control or pro-calcific diet for 16 weeks and monitored via echocardiography, histology, immunohistochemistry, and quantitative polarized light imaging. Additionally, TPEF imaging was used to quantify tissue autofluorescence (A) at 755 nm, 810 nm and 860 nm excitation, to calculate TPEF 755-860 ratio (A
/(A
+ A
)) and TPEF Collagen-Calcium ratio (A
/(A
+ A
)) in the murine valves. In a separate experiment, animals were fed the above diets till 28 weeks to assess for later-stage calcification.
Pro-calcific mice showed evidence of lipid deposition at 4 weeks and calcification at 16 weeks at the valve commissures. The valves of pro-calcific mice also showed positive expression for markers of osteogenic differentiation, myofibroblast activation, proliferation, inflammatory cytokines and collagen remodeling. Pro-calcific mice exhibited lower TPEF autofluorescence ratios, at locations coincident with calcification, that correlated with increased collagen disorganization and positive expression of osteogenic markers. Additionally, locations with lower TPEF autofluorescence ratios at 4 and 16 weeks exhibited increased calcification at later 28-week timepoints.
This study suggests the potential of TPEF autofluorescence metrics to serve as a label-free tool for early detection and monitoring of CAVD pathophysiology.
Tissue engineering using scaffolds and growth factors is a crucial approach in bone regeneration and repair. The combination of bioactive agents carrying microparticles with porous scaffolds can be ...an efficient solution when controlled release of bio-signalling molecules is required. The present study was based on a recent approach using a biodegradable scaffold and protein-loaded microparticles produced in an innovative manner in which protein loss is minimized during the loading process. Bovine serum albumin (BSA)-loaded gelatin microparticles were obtained by grinding freeze-dried membranes of gelatin and BSA. Porous scaffolds (250-355 µm pore size) produced from a polyactide (PLLA) and polycaprolactone (PCL) blend by salt leaching/supercritical CO₂ methods were used for the experiments. Gelatin microparticles containing three different BSA amounts were incorporated into the porous scaffolds by using a surfactant. In vitro release profiles showed up to 90% protein loading efficiency. This novel method appears to be an effective approach for producing particles that can minimize protein loss during the loading process.
Statement of Purpose
All implanted materials induce an immune response driven by macrophages called the foreign body reaction (FBR). Macrophages exist in a continuum of highly inflammatory to highly ...pro‐wound healing phenotypic cell types. This project aims to assess macrophage activation patterns caused by implanted polyvinyl alcohol non‐biodegradable vs. collagen biodegradable sponges in the subcutaneous space of male Sprague Dawley rats. Our hypothesis is that macrophages interact differently between different materials. If such difference exists, this means the approaches to driving macrophages to their wound healing states would be expected to be different. This study included the delivery of previously tested drug modulators, dexamethasone and iloprost, for their ability to shift macrophages to their pro‐wound healing state. The use of these modulators enables screening and comparing the effect of biomaterial chemistry on macrophage activation patterns.
Methods
Male Sprague‐Dawley rats, (250 – 290) grams, were divided into three groups (6 animals in groups #1 and #2 and 4 animals in group #3) where PVA sponges were soaked in either the vehicle solution (lactate Ringers' solution) in group #1 (control group), 100 μM dexamethasone in group #2 (treatment group), or 1 nM iloprost in group #3 (treatment group). Two PVA sponges were implanted in the dorsal subcutaneous space of each animal. A daily booster dose of the assigned treatment was administered subcutaneously into each implantation site 24 hours after implantation until experiment completion. A week after implantation, sponges and surrounding tissue were harvested for either histological/immunohistochemical analysis or CCL2 quantification.
Results
C‐C motif chemokine ligand 2 (CCL2) levels averaged around 800, 450, and 300 ng/mL in the wound fluid collected from the control, iloprost treated, and dexamethasone‐treated sponges, respectively. The levels of CCL2 in the wound fluid collected from the dexamethasone‐treated sponges and those collected from the iloprost treated sponges were found to be significantly less than the wound fluid levels of CCL2 from the control sponges. No significant difference was found between the wound fluid levels of CCL2 from dexamethasone‐treated sponges vs. iloprost‐treated sponges (Figure 1).
Less cellular density and less collagen formation were observed in the tissues surrounding both iloprost treated sponges and dexamethasone‐treated sponges compared to the tissues around the control sponges (Figure 2).
The total protein concentrations in the wound fluid collected from control and treatment sponges were similar within each animal and ranged between 40–50 mg/mL.
Additional experiments are needed to determine the macrophage activation response after the implantation of collagen bio‐degradable sponges and compare it to macrophage activation response after the implantation of PVA non‐biodegradable sponges.
Support or Funding Information
Provost's Collaborative Research Grant at University of Arkansas
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
Cardiac valves are sophisticated, dynamic structures residing in a complex mechanical and hemodynamic environment. Cardiac valve disease is an active and progressive disease resulting in severe ...socioeconomic burden, especially in the elderly. Valve disease also leads to a 50% increase in the possibility of associated cardiovascular events. Yet, valve replacement remains the standard of treatment with early detection, mitigation, and alternate therapeutic strategies still lacking. Effective study models are required to further elucidate disease mechanisms and diagnostic and therapeutic strategies. Organ-on-chip models offer a unique and powerful environment that incorporates the ease and reproducibility of
systems along with the complexity and physiological recapitulation of the
system. The key to developing effective valve-on-chip models is maintaining the cell and tissue-level microenvironment relevant to the study application. This review outlines the various components and factors that comprise and/or affect the cell microenvironment that ought to be considered while constructing a valve-on-chip model. This review also dives into the advancements made toward constructing valve-on-chip models with a specific focus on the aortic valve, that is,
studies incorporating three-dimensional co-culture models that incorporate relevant extracellular matrices and mechanical and hemodynamic cues.
DL-3-n-butylphthalide (DL-NBP) is one of the constituents of Chinese celery extract that is used to treat stroke, dementia, and ischemic diseases. However, its role in traumatic brain injury is less ...well known. In this investigation, neuroprotective effects of DL-NBP in concussive head injury (CHI) on brain pathology were explored in a rat model. CHI was inflicted in anesthetized rats by dropping a weight of 114.6 g from a height of 20 cm through a guide tube on the exposed right parietal bone inducing an impact of 0.224 N and allowed them to survive 4 to 24 h after the primary insult. DL-NBP was administered (40 or 60 mg/kg, i.p.) 2 and 4 h after injury in 8-h survival group and 8 and 12 h after trauma in 24-h survival group. In addition, TiO
2
-nanowired delivery of DL-NBP (20 or 40 mg/kg, i.p.) in 8 and 24 h CHI rats was also examined. Untreated CHI showed a progressive increase in blood-brain barrier (BBB) breakdown to Evans blue albumin (EBA) and radioiodine (
131-
I), edema formation, and neuronal injuries. The magnitude and intensity of these pathological changes were most marked in the left hemisphere. Treatment with DL-NBP significantly reduced brain pathology in CHI following 8 to 12 h at 40-mg dose. However, 60-mg dose is needed to thwart brain pathology at 24 h following CHI. On the other hand, TiO
2
-DL-NBP was effective in reducing brain damage up to 8 or 12 h using a 20-mg dose and only 40-mg dose was needed for neuroprotection in CHI at 24 h. These observations are the first to suggest that (i) DL-NBP is quite effective in reducing brain pathology and (ii) nanodelivery of DL-NBP has far more superior effects in CHI, not reported earlier.
Neprilysin (NPL), the rate-limiting enzyme for amyloid beta peptide (AβP), appears to play a crucial role in the pathogenesis of Alzheimer’s disease (AD). Since mesenchymal stem cells (MSCs) and/or ...cerebrolysin (CBL, a combination of neurotrophic factors and active peptide fragments) have neuroprotective effects in various CNS disorders, we examined nanowired delivery of MSCs and CBL on NPL content and brain pathology in AD using a rat model. AD-like symptoms were produced by intraventricular (i.c.v.) administration of AβP (1-40) in the left lateral ventricle (250 ng/10 μl, once daily) for 4 weeks. After 30 days, the rats were examined for NPL and AβP concentrations in the brain and related pathology. Co-administration of TiO2-nanowired MSCs (10
6
cells) with 2.5 ml/kg CBL (i.v.) once daily for 1 week after 2 weeks of AβP infusion significantly increased the NPL in the hippocampus (400 pg/g) from the untreated control group (120 pg/g; control 420 ± 8 pg/g brain) along with a significant decrease in the AβP deposition (45 pg/g from untreated control 75 pg/g; saline control 40 ± 4 pg/g). Interestingly, these changes were much less evident when the MSCs or CBL treatment was given alone. Neuronal damages, gliosis, and myelin vesiculation were also markedly reduced by the combined treatment of TiO2, MSCs, and CBL in AD. These observations are the first to show that co-administration of TiO2-nanowired CBL and MSCs has superior neuroprotective effects in AD probably due to increasing the brain NPL level effectively, not reported earlier.
The possibility that traumatic brain injury (TBI) occurring in a cold environment exacerbates brain pathology and oxidative stress was examined in our rat model. TBI was inflicted by making a ...longitudinal incision into the right parietal cerebral cortex (2 mm deep and 4 mm long) in cold-acclimatized rats (5 °C for 3 h daily for 5 weeks) or animals at room temperature under Equithesin anesthesia. TBI in cold-exposed rats exhibited pronounced increase in brain lucigenin (LCG), luminol (LUM), and malondialdehyde (MDA) and marked pronounced decrease in glutathione (GTH) as compared to identical TBI at room temperature. The magnitude and intensity of BBB breakdown to radioiodine and Evans blue albumin, edema formation, and neuronal injuries were also exacerbated in cold-exposed rats after injury as compared to room temperature. Nanowired delivery of H-290/51 (50 mg/kg) 6 and 8 h after injury in cold-exposed group significantly thwarted brain pathology and oxidative stress whereas normal delivery of H-290/51 was neuroprotective after TBI at room temperature only. These observations are the first to demonstrate that (i) cold aggravates the pathophysiology of TBI possibly due to an enhanced production of oxidative stress, (ii) and in such conditions, nanodelivery of antioxidant compound has superior neuroprotective effects, not reported earlier.
The possibility that stress associated with chronic forced swim (FS) may exacerbate methamphetamine (METH) neurotoxicity was examined in a rat model. Rats were subjected to FS in a pool (30 °C) for ...15 min daily for 8 days. Control rats were kept at room temperature. METH was administered (9 mg/kg, s.c.) in both control and FS rats and allowed to survive 4 h after the drug injection. METH in FS rats exacerbated BBB breakdown to Evans blue albumin (EBA) by 150 to 220% and
131
-Iodine by 250 to 380% as compared to naive rats after METH. The METH-induced BBB leakage was most pronounced in the cerebral cortex followed by the hippocampus, cerebellum, thalamus, and hypothalamus in both FS and naive rats. The regional BBB changes were associated with a reduction in the local cerebral blood flow (CBF). Brain edema was also higher by 2 to 4% in FS rats after METH than in naive animals. Neuronal and glial cell injuries were aggravated by threefold to fivefold after METH in FS than the control group. Pretreatment with ondansetron (1 mg/kg, i.p.) 30 min before METH injection in naive rats reduced the brain pathology and improved the CBF. However, TiO
2
-nanowired delivery of ondansetron (1 mg/kg, i.p.) was needed to reduce METH-induced brain damage, BBB leakage, reduction in CBF, and edema formation in FS. Taken together, these observations are the first to show that METH exacerbates BBB breakdown leading to neurotoxicity in FS animals. This effect of METH-induced BBB breakdown and brain pathology in naive and FS rats is attenuated by ondansetron treatment indicating an involvement of 5-HT3 receptors, not reported earlier.
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