Sex-Dependent Impairment of Endothelium-Dependent Relaxation in Aorta of Mice with Overexpression of Hyaluronan in Tunica Media.

Diabetic macroangiopathy is characterized by increased extracellular matrix deposition, including excessive hyaluronan accumulation, vessel thickening and stiffness, and endothelial dysfunction in large arteries. We hypothesized that the overexpression of hyaluronan in the tunica media also led to endothelial cell (EC) dysfunction. To address this hypothesis, we investigated the following in the aortas of mice with excessive hyaluronan accumulation in the tunica media (HAS-2) and wild-type mice: EC dysfunction via myograph studies, nitric oxide (NO) bioavailability via diaminofluorescence, superoxide formation via dihydroethidium fluorescence, and the distances between ECs via stereological methods. EC dysfunction, characterized by blunted relaxations in response to acetylcholine and decreased NO bioavailability, was found in the aortas of male HAS-2 mice, while it was unaltered in the aortas of female HAS-2 mice. Superoxide levels increased and extracellular superoxide dismutase (ecSOD) expression decreased in the aortas of male and female HAS-2 mice. The EC-EC distances and LDL receptor expression were markedly increased in the HAS-2 aortas of male mice. Our findings suggest hyaluronan increases oxidative stress in the vascular wall and that together with increased EC distance, it is associated with a sex-specific decrease in NO levels and endothelial dysfunction in the aorta of male HAS-2 transgenic mice.

Electrospun nanofiber mesh with connective tissue growth factor and mesenchymal stem cells for pelvic floor repair: Long-term study.

Pelvic organ prolapse (POP) affects many women, with an estimated lifetime risk of surgical intervention of 18.7%. There is a need for alternative approaches as the use of synthetic nondegradable mesh was stopped due to severe adverse events, and as current methods for pelvic floor repair have high POP recurrence rates. Thus, we hypothesized that electrospun degradable meshes with stem cells and growth factor were safe and durable for the long term in elderly rats. In an abdominal repair model, electrospun polycaprolactone (PCL) meshes coated with connective tissue growth factor (CTGF)/PEG-fibrinogen (PF) and rat mesenchymal stem cells were implanted in elderly female rats and removed after in average 53 weeks (53-week group). Collagen amount and production were quantified by qPCR and Western blotting. Moreover, histological appearance and biomechanical properties were evaluated. Results were compared with previous results of young rats with identical mesh implanted for 24 weeks (24-week group). The 53-week group differed from the 24-week group in terms of (1) reduced collagen III, (2) strong reduction in foreign body response, and (3) altered histological appearance. We found comparable biomechanical properties, aside from higher, not significant, mean tissue stiffness in the 53-week group. Lastly, we identified mesh components 53 weeks after implantation. This study provides new insights into future POP repair in postmenopausal women by showing how CTGF/PF-coated electrospun PCL meshes with stem cells exhibit sufficient support, biocompatibility, and no mesh-related complications long term in an abdominal repair model in elderly rats.

Increased activity of the metalloproteinase PAPP-A promotes diabetes-induced glomerular hypertrophy.

BACKGROUND: Diabetic nephropathy (DN) is a serious complication of diabetes and a common cause of end stage renal failure. Insulin-like growth factor (IGF)-signaling has been implicated in DN, but is mechanistically poorly understood. Here, we assessed the activity of the metalloproteinase PAPP-A, an activator of IGF activity, and its possible interaction with the endogenous PAPP-A inhibitors stanniocalcin (STC)-1 and -2 in the mammalian kidney under normal and hyperglycemic conditions. METHODS AND RESULTS: Immunohistochemistry demonstrated that PAPP-A, its proteolytic substrate IGF binding protein-4, STC1 and STC2 are present in the human kidney. Endogenous inhibited complexes of PAPP-A (PAPP-A:STC1 and PAPP-A:STC2) were demonstrated in media conditioned by human mesangial cells (HMCs), suggesting that PAPP-A activity is regulated by the STCs in kidney tissue. A method for the selective detection of active PAPP-A in tissue was developed and a significant increase in glomerular active PAPP-A in human diabetic kidney relative to normal was observed. In DN patients, the estimated glomerular filtration rate correlated with PAPP-A activity. In diabetic mice, glomerular growth was reduced when PAPP-A activity was antagonized by adeno-associated virus-mediated overexpression of STC2. CONCLUSION: We propose that PAPP-A activity in renal tissue is precisely balanced by STC1 and STC2. An imbalance in this equilibrium causing increased PAPP-A enzymatic activity potentially contributes to the development of DN, and thus, therapeutic targeting of PAPP-A activity may represent a novel strategy for its treatment.

Electrospun nanofiber mesh with fibroblast growth factor and stem cells for pelvic floor repair.

Surgical outcome following pelvic organ prolapse (POP) repair needs improvement. We suggest a new approach based on a tissue-engineering strategy. In vivo, the regenerative potential of an electrospun biodegradable polycaprolactone (PCL) mesh was studied. Six different biodegradable PCL meshes were evaluated in a full-thickness abdominal wall defect model in 84 rats. The rats were assigned into three groups: (1) hollow fiber PCL meshes delivering two dosages of basic fibroblast growth factor (bFGF), (2) solid fiber PCL meshes with and without bFGF, and (3) solid fiber PCL meshes delivering connective tissue growth factor (CTGF) and rat mesenchymal stem cells (rMSC). After 8 and 24 weeks, we performed a histological evaluation, quantitative analysis of protein content, and the gene expression of collagen-I and collagen-III, and an assessment of the biomechanical properties of the explanted meshes. Multiple complications were observed except from the solid PCL-CTGF mesh delivering rMSC. Hollow PCL meshes were completely degraded after 24 weeks resulting in herniation of the mesh area, whereas the solid fiber meshes were intact and provided biomechanical reinforcement to the weakened abdominal wall. The solid PCL-CTGF mesh delivering rMSC demonstrated improved biomechanical properties after 8 and 24 weeks compared to muscle fascia. These meshes enhanced biomechanical and biochemical properties, demonstrating a great potential of combining tissue engineering with stem cells as a new therapeutic strategy for POP repair. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:48-55, 2020.

Electrospun biodegradable microfibers induce new collagen formation in a rat abdominal wall defect model: A possible treatment for pelvic floor repair?

Half of the female population over age 50 years will experience pelvic organ prolapse. We suggest a new approach based on tissue engineering principles to functionally reconstruct the anatomical structures of the pelvic floor. The aim of this study is to investigate the mechanical performance and effect on collagen and elastin production of a degradable mesh releasing basic fibroblast growth factor (bFGF). Implantation of biodegradable mesh with or without bFGF in their core has been conducted in 40 rats in an abdominal wall defect model. Samples were explanted after 4, 8, and 24 weeks, and tested for mechanical properties and the composition of connective tissue. The study showed an increase in mRNA expression for collagen-I (p = 0.0060) and collagen-III (p = 0.0086) in the 4 weeks group with bFGF. The difference was equalized at 8 and 24 weeks. No difference was found at any time for protein amount for collagen-I, collagen-III, and fibronectin. The amount of collagen decreased from 4 to 24 weeks but the fraction of collagen increased. The maximal load of the newly formed tissue showed no effect of bFGF at any time. Exclusively, histology showed a limited ingrowth of collagen fibers after 4 weeks with bFGF but signs of elastin fibers were seen at 24 weeks. The investigation showed that a biodegradable mesh promotes tissue formation with a promising strength. The mesh with bFGF did not represent any advantage on either long or short term in comparison to the mesh without bFGF. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 680-688, 2018.

Fibrogenic and angiogenic commitments of human induced pluripotent stem cells derived mesenchymal stem cells in connective tissue growth factor-delivering scaffold in an immune-deficient mice model.

Compared to terminal differentiated cells, stem cells play important roles in the maintenance and regeneration, and thus have been intensively researched as the most promising cell based therapy. In order to maximize the effectiveness of stem cell based therapies, it is essential to understand the environmental (niche) signals that regulate stem cell behavior. Recent findings suggest that fibroblasts have a mesenchymal origin and that mesenchymal stem cells (MSCs) demonstrate proangiogenic function, where both fibrogenic and angiogenic activities are associated with connective tissue growth factor (CTGF), a matricellular protein that serves as an essential mediator of skeletogenesis in development and vascular remodeling. Here, for the first time, we demonstrate that upon local delivery of CTGF from a three dimensional (3D) nanocomposite scaffold, human induced pluripotent stem cells derived MSCs can be directed to differentiate toward fibroblasts in a 3D nanocomposite scaffold in female nonobese diabetic CB-17/Icr-severe combined immunodeficient mice. The stem cell-scaffold constructs present not only intriguingly strong fibroblastic commitments but also angiogenic induction in vivo. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2266-2274, 2018.

The employers' perspective on how PhD training affects physicians' performance in the clinic.

INTRODUCTION: An increase in the number of MD-PhDs has sparked debate as to how physicians with PhD research training contribute in the clinic. This study focuses on the development and employment situation of MD-PhDs from Aarhus University, Denmark, and on the impact of MD-PhDs in the clinic as seen from the employers' perspective. METHODS: The study is based on a mixed methods approach using both quantitative and qualitative data. Quantitative data hail from existing statistical data and reports, while the qualitative data stem from semi-structured interviews with six executive consultants and 36 members of appointment committees, mainly from hospitals in the Central Denmark Region. RESULTS: Quantitative data reveal an increase in the number of MD-PhDs concluding their training at Aarhus University. The MD-PhDs are employed in the public sector and, overall, their skills match the employers' demands. Qualitative data show that employers were satisfied with the skills the MD-PhDs brought to the clinic, particularly in terms of their ability to assess and use new and relevant information and to instigate a more scientific approach in the clinic. Informants from remote hospitals expressed a demand for more MD-PhDs, while other informants were concerned about how some MD-PhDs stopped doing medical research in the clinic after completing their PhD. CONCLUSION: Overall, employers seem satisfied with the skills that MD-PhDs bring to the clinic. However, some voice concern that too much importance is attached to the PhD degree and that some MD-PhDs are not active doing research. FUNDING: The study was funded by the Graduate School of Health (Aarhus University) and The Central Denmark Region. TRIAL REGISTRATION: not relevant.

Renal PET-imaging with (11)C-metformin in a transgenic mouse model for chronic kidney disease.

BACKGROUND: Organic cation transporters (OCTs) in the renal proximal tubule are important for the excretion of both exo- and endogenous compounds, and chronic kidney disease (CKD) alter the expression of OCT. Metformin is a well-known substrate for OCT, and recently, we demonstrated that positron emission tomography (PET) with 11C-labelled metformin ((11)C-metformin) is a promising approach to evaluate the function of OCT. The aim of this study is therefore to examine renal pharmacokinetics of (11)C-metformin and expression of OCTs in a transgenic (RenTGF-ß1) mouse model of CKD. METHODS: Age- and sex-matched RenTGF-ß1 (Tg) and wildtype (WT) mice were used (5-8/group). Animals received an iv bolus of (11)C-metformin followed by 90-min dynamic PET and MRI scan. PET data were analysed using a one-tissue compartment model. Renal protein abundance of OCT2 (by Western blot) as well as OCT1, OCT2, and MATE1 messenger RNA (mRNA) (by RT-PCR) was examined. RESULTS: Protein expression of the basolateral uptake transporter OCT2 was 1.5-fold lower in Tg mice compared to WT mice while OCT1 and MATE1 mRNA expression did not differ between the two groups. The influx rate constant of (11)C-metformin in renal cortex (K 1) was 2.2-fold lower in transgenic mice whereas the backflux rate constant (k 2) was similar in the two groups, consistent with protein expression. Total body clearance (TBC) correlated within each group linearly with K 1. CONCLUSIONS: In conclusion, this study demonstrates that both renal OCT2 expression and (11)C-metformin uptake are reduced in CKD mice. This potentially makes (11)C-metformin valuable as a PET probe to evaluate kidney function.

Mechanisms involved in extracellular matrix remodeling and arterial stiffness induced by hyaluronan accumulation.

BACKGROUND AND AIMS: Hyperglycemia induces hyaluronan (HA) accumulation in the vasculature. Excessive accumulation of HA around the vascular smooth muscle cells (VSMC) results in increased aortic stiffness and strength and accelerated atherosclerosis in ApoE(-)/(-) mice. We hypothesized that HA accumulation primes the vasculature for atherosclerosis by crosslinking and reorganizing the extracellular matrix (ECM) and by pushing VSMC differentiation towards a less mature phenotype. METHODS: Aortas from HAS-2 transgenic (Tg) mice and wild type mice were used for all experiments. Biomechanics and cross-sectional area measurements were performed before and after HA digestion. The vessel and ECM composition was examined by immunoblotting and electron microscopy. Primary VSMC cultures were examined by qPCR and thymidine incorporation. RESULTS: Tg mice aorta cross-sectional area was increased before (14%, p = 0.0148), but not after HA digestion (p = 0.3437). The increase in vessel stiffness (32%, p = 0.0217) and strength (31%, p = 0.0043) in the Tg aorta persisted after HA digestion. Crosslinking of HA by heavy chains from Inter-α-Inhibitor was increased (175%, p = 0.0006). The Tg VSMCs have the appearance of a synthetic phenotype supported by a 40% decrease in α-smooth muscle actin isoform X1 (p = 0.0296) and an increase in proliferation (63%, p = 0.0048) and osteoprotegerin production (133%, p = 0.0010) in cultured Tg VSMCs. CONCLUSIONS: Our results show that induced HA accumulation is followed by increased HA crosslinking and create a shift in VSMC phenotype and proliferation. These findings may provide a mechanism for how hyperglycemia through HA accumulation prime the vascular wall for cholesterol and leucocyte accumulation and development of atherosclerosis.

Restoration of Haemoglobin Level Using Hydrodynamic Gene Therapy with Erythropoietin Does Not Alleviate the Disease Progression in an Anaemic Mouse Model for TGFß1-Induced Chronic Kidney Disease.

Erythropoietin, Epo, is a 30.4 kDa glycoprotein hormone produced primarily by the fetal liver and the adult kidney. Epo exerts its haematopoietic effects by stimulating the proliferation and differentiation of erythrocytes with subsequent improved tissue oxygenation. Epo receptors are furthermore expressed in non-haematopoietic tissue and today, Epo is recognised as a cytokine with many pleiotropic effects. We hypothesize that hydrodynamic gene therapy with Epo can restore haemoglobin levels in anaemic transgenic mice and that this will attenuate the extracellular matrix accumulation in the kidneys. The experiment is conducted by hydrodynamic gene transfer of a plasmid encoding murine Epo in a transgenic mouse model that overexpresses TGF-ß1 locally in the kidneys. This model develops anaemia due to chronic kidney disease characterised by thickening of the glomerular basement membrane, deposition of mesangial matrix and mild interstitial fibrosis. A group of age matched wildtype littermates are treated accordingly. After a single hydrodynamic administration of plasmid DNA containing murine EPO gene, sustained high haemoglobin levels are observed in both transgenic and wildtype mice from 7.5 ± 0.6 mmol/L to 9.4 ± 1.2 mmol/L and 10.7 ± 0.3 mmol/L to 15.5 ± 0.5 mmol/L, respectively. We did not observe any effects in the thickness of glomerular or tubular basement membrane, on the expression of different collagen types in the kidneys or in kidney function after prolonged treatment with Epo. Thus, Epo treatment in this model of chronic kidney disease normalises haemoglobin levels but has no effect on kidney fibrosis or function.

Expression of hypoxia-inducible factor-1α and hepatocyte growth factor in development of fibrosis in the transplanted kidney.

Late renal graft loss is associated with interstitial fibrosis. Hypoxia-inducible factor-1α (HIF-1α) is thought to facilitate fibrosis through interaction with TGF-ß1, while hepatocyte growth factor (HGF) may act antifibrotic in the kidney allograft. The aim of this study was to investigate the expression of HIF-1α and HGF in protocol biopsies as possible prognostic biomarkers for renal fibrosis. Thirty-nine renal transplant recipients were included in the study. Protocol biopsies performed 1 and 2 years after transplantation were used for immunohistochemistry analysis. The correlation between HIF-1α/HGF and the Banff score was analysed. In addition, progression in renal fibrosis and graft survival among recipients with high or low expression of HIF-1α/HGF after transplantation was compared. There was no significant correlation between fibrosis and the HIF-1α expression 1 and 2 years after transplantation, but an inverse significant correlation between the HGF expression and the fibrosis score 1 year after transplantation was shown. Even when adjusting for human leucocyte antigen mismatches, there was a significant relationship between fibrosis and HGF expression. Graft survival was not significantly correlated to HIF-1α or HGF at 1 year, although the trend was towards better graft survival with high HGF. HGF may have antifibrotic effects in human renal transplants. (Central.Denmark.Region.Committee number: 1-10-72-318-13).

No effect of remote ischaemic conditioning on inflammation in a porcine kidney transplantation model.

MAIN PROBLEM: Delayed graft function after kidney transplantation is associated with decreased graft survival and increased patient mortality but the pathogenesis is poorly understood. Remote ischaemic conditioning (rIC) may prevent delayed graft function by an anti-inflammatory effect. In a porcine model of transplantation from adults to children, we investigated the inflammatory response in the transplanted kidney and the effect of rIC. METHODS: Kidneys were recovered from brain dead donor pigs(63kg) and transplanted into two groups of recipient pigs(15kg) after 22h of cold ischaemia. Recipients were randomised to either: rIC (n=8) performed before the 10-h reperfusion period or no-rIC (n=8). Non-transplanted kidneys from eight brain dead pigs served as controls. RESULTS: Compared to controls, transplantation increased the number of apoptotic cells, macrophages and neutrophils in the kidney. After transplantation, IL-10 levels increased and IL-6 levels decreased in the kidney, whereas levels of TNF-α and IL-8 were not affected. A significant rise in plasma IL-1ß and IL-6 was observed in the recipients after transplantation. Plasma IL-10 was not affected by transplantation and TNF-α and IL-8 were below detection limit. No effect of rIC was found with regards to cell infiltration or cytokine production. CONCLUSION: Renal transplantation elicits an inflammatory response in the kidney manifested as apoptotic cell death, macrophage and neutrophil infiltration, and an anti-inflammatory cytokine response 10h after transplantation. This response was not modified by rIC.

The effects of adenocaine (adenosine and lidocaine) on early post-resuscitation cardiac and neurological dysfunction in a porcine model of cardiac arrest.

AIM: Return of spontaneous circulation (ROSC) elicits ischaemia/reperfusion injury and myocardial dysfunction. The combination of adenosine and lidocaine (AL, adenocaine) has been shown to (1) inhibit neutrophil inflammatory activation and (2) improve left ventricular function after ischaemia. We hypothesized that resuscitation with adenocaine during early moments of cardiopulmonary resuscitation (CPR) attenuates leucocyte oxidant generation and myocardial dysfunction. METHODS: Pigs were randomized to: (1) sham (n=7), (2) cardiac arrest (CA; n=16), or 3) cardiac arrest+adenocaine (CA+AL; n=12). After 7 min of electrically induced ventricular fibrillation, start of CPR was followed by infusion of saline (CA) or adenocaine (CA+AL) for 6 min. Haemodynamics, cardiodynamics (pressure-volume loops) and leucocyte superoxide anion generation were assessed. Neurological function was evaluated after 24h by histology and neurological deficit score (0=normal; 500=brain dead). RESULTS: Rate of ROSC was comparable between groups: CA group 11/16 and CA+AL group 7/12 p=0.57). Cardiac index transiently increased after ROSC in both groups. Left ventricular dysfunction demonstrated by a rightward shift of the intercept of end-systolic pressure-volume relations in CA was avoided in the CA+AL group. Leucocyte superoxide anion generation 2h after ROSC was significantly attenuated in the CA+AL group compared to the CA group. Neurological deficit scores [CA: median: 17.5(IQR:0-75) and CA+AL: 35(IQR:15-150)] and histopathological damage were comparable in both groups (p=0.37). CONCLUSION: Infusion of adenocaine during early resuscitation from CA significantly improved early post-resuscitation cardiac function and attenuated leucocyte superoxide anion generation, without a change in post-ROSC neurological function. (IACUC protocol number 023-2009).

Soluble adhesion molecules correlate with surface expression in an in vitro model of endothelial activation.

Endothelial activation is a pivotal event in the development and progression of inflammation. Central to endothelial activation is the up-regulation of cellular adhesion molecules (CAMs) including E-selectin (CD62E), ICAM-1 (CD54), VCAM-1 (CD106) and PECAM-1 (CD31). These CAMs are also found in soluble forms (sCAMs). In this in vitro study of endothelial activation, we examined whether the levels of sCAMs correlate with the endothelial surface expression of CAMs in a dose-dependent and time-dependent manner. Such a correlation would support the use of sCAMs as surrogate markers for endothelial activation in inflammatory conditions. Human umbilical vein endothelial cells (HUVEC) were cultured with various concentrations of TNF-α for 8 hr and at a fixed concentration of TNF-α for various durations. The levels of soluble and surface-bound E-selectin, ICAM-1, VCAM-1 and PECAM-1 were quantified by flow cytometry. TNF-α stimulation increased CAM and sCAM expression in a dose-dependent and time-dependent manner. There was a significant positive correlation between the levels of ICAM-1 and sICAM-1 and between the levels of VCAM and sVCAM-1 in both the dose-response and time-response experiments. A positive correlation between the levels of E-selectin and sE-selectin was observed in the time-response experiment. This study supports the use of sCAMs as potential biomarkers of endothelial activation. In particular, the use of sICAM-1, sVCAM-1 and sE-selectin seems promising.

SKG arthritis as a model for evaluating therapies in rheumatoid arthritis with special focus on bone changes.

The aim was to further characterize the SKG model of rheumatoid arthritis (RA) and its potential for studying intervention treatments, with special focus on bone targeting therapies. Three individual studies were conducted, using a total of 71 SKG mice, comparing arthritis induction with mannan versus zymosan A, female versus male mice, and the effect of dexamethasone intervention treatment initiated at different time points after arthritis induction. Hind paws were embedded undecalcified in methyl methacrylate, and sections were stained with Masson-Goldner trichrome. Areal Bone Mineral Density (aBMD) of the femora was determined with pDXA. RNA was extracted from the hind paws followed by the quantification by reverse transcriptase PCR. SKG mice stimulated with mannan presented a higher arthritis score than mice stimulated with zymosan A. Female SKG mice developed a more severe arthritis than male SKG mice. Dexamethasone inhibited arthritis clinically as well as histologically when the treatment was initiated prophylactically or within the first week of arthritis. Femoral aBMD was lower in animals with arthritis than in control animals. The RANKL RNA expression was elevated in arthritic mice, whereas OPG RNA expression was unchanged. The results suggest mannan as arthritis inductor and female instead of male mice in experiments as well as an optimal time window for the initiation of treatment. Systemic bone loss as well as local up regulation of RANKL was present early in SKG arthritis. These results demonstrate that SKG arthritis is a suitable new model for evaluating therapies in RA.

Differential effects of Smad3 targeting in a murine model of chronic kidney disease.

Transforming growth factor (TGF)-ß1 has a pivotal role in the pathogenesis of progressive kidney diseases that are characterized by fibrosis. The main intracellular signaling pathway of TGF-ß1 is the Smad system, where Smad2 and Smad3 play a central role in transcriptional regulation of target genes involved in extracellular matrix (ECM) metabolism. This study analyzes the hypothesis that blockade of Smad3 attenuates the development of TGF-ß1-driven renal fibrosis. This was examined in vivo in a transgenic model of TGF-ß1-induced chronic kidney disease with Smad3 or without Smad3 expression and in vitro in mesangial cells and glomerular endothelial cells with Smad2/3 inhibitors or Smad3-knockdown. Electron microscopy was used for evaluation of morphological changes, real-time polymerase chain reaction for detection of RNA expression, and immunohistochemistry for localization of ECM components. Matrix metalloproteinase (MMP) level was assessed by gelatin zymography electrophoresis and located by in situ zymography. The results show TGF-ß1-induced mesangial matrix expansion, tubulointerstitial fibrosis, and tubular basement membrane thickening that are attenuated by Smad3 deletion, whereas TGF-ß1-induced glomerular basement membrane thickening is not shown. The amount and distribution profile of MMP-2 may suggest a role of the enzyme herein. We conclude that Smad3 targeting is not exclusively beneficial as Smad3 has diverse transcriptional regulatory effects in different cell types in the kidney.

Adenocaine and Mg(2+) reduce fluid requirement to maintain hypotensive resuscitation and improve cardiac and renal function in a porcine model of severe hemorrhagic shock*.

OBJECTIVES: Hypotensive resuscitation is gaining clinical acceptance in the treatment of hemorrhagic shock. Our aims were to investigate: 1) the effect of 7.5% NaCl with adenocaine (adenosine and lidocaine, AL) and AL with Mg (ALM) on fluid requirement to maintain a minimum mean arterial pressure of 50 mm Hg, and 2) the effect of a second bolus of 0.9% NaCl with AL during return of shed blood on cardiac and renal function in a porcine model of hemorrhagic shock. DESIGN: Pigs were randomized to: Sham (n = 5), Sham + ALM/AL (n = 5), hemorrhage control (n = 11), or hemorrhage + ALM/AL (n = 9). Hemorrhage animals were bled to a mean arterial pressure of 35 mm Hg. After 90 mins, pigs were fluid resuscitated with Ringers acetate and 20 mL 7.5% NaCl with ALM to maintain a target mean arterial pressure of minimum 50 mm Hg. Shed blood and 0.9% NaCl with AL were infused 30 mins later. Hemorrhage control group was subjected to the same protocol but without ALM/AL. Hemodynamics, cardiodynamics (pressure-volume analysis), oxygen consumption, and kidney function were measured for 6 hrs. SETTING: University hospital laboratory. SUBJECTS: Female farm-bred pigs. RESULTS: Fluid volume infused during hypotensive resuscitation was 40% less in the 7.5% NaCl-/ALM-treated pigs than controls (25 vs. 41 mL/kg, p < .05). ALM was associated with a significant increase in dp/dtmax, end-systolic blood pressure, and systemic vascular resistance. Return of shed blood and 0.9% NaCl/AL reduced whole body oxygen consumption by 27% (p < .05), and significantly improved the end-systolic pressure-volume relationship and preload recruitable stroke work compared to controls. Glomerular filtration rate in the ALM/AL group returned to 83% of baseline compared to 54% in controls (p = .01). CONCLUSION: Resuscitation with 7.5% NaCl ALM increases cardiac function and reduces fluid requirements during hypotensive resuscitation, whereas a second AL infusion during blood resuscitation transiently reduces whole body oxygen consumption and improves cardiac and renal function.

Unique structural features facilitate lizard tail autotomy.

Autotomy refers to the voluntary shedding of a body part; a renowned example is tail loss among lizards as a response to attempted predation. Although many aspects of lizard tail autotomy have been studied, the detailed morphology and mechanism remains unclear. In the present study, we showed that tail shedding by the Tokay gecko (Gekko gecko) and the associated extracellular matrix (ECM) rupture were independent of proteolysis. Instead, lizard caudal autotomy relied on biological adhesion facilitated by surface microstructures. Results based on bio-imaging techniques demonstrated that the tail of Gekko gecko was pre-severed at distinct sites and that its structural integrity depended on the adhesion between these segments.

Severe lung fibrosis requires an invasive fibroblast phenotype regulated by hyaluronan and CD44.

Tissue fibrosis is a major cause of morbidity, and idiopathic pulmonary fibrosis (IPF) is a terminal illness characterized by unremitting matrix deposition in the lung. The mechanisms that control progressive fibrosis are unknown. Myofibroblasts accumulate at sites of tissue remodeling and produce extracellular matrix components such as collagen and hyaluronan (HA) that ultimately compromise organ function. We found that targeted overexpression of HAS2 (HA synthase 2) by myofibroblasts produced an aggressive phenotype leading to severe lung fibrosis and death after bleomycin-induced injury. Fibroblasts isolated from transgenic mice overexpressing HAS2 showed a greater capacity to invade matrix. Conditional deletion of HAS2 in mesenchymal cells abrogated the invasive fibroblast phenotype, impeded myofibroblast accumulation, and inhibited the development of lung fibrosis. Both the invasive phenotype and the progressive fibrosis were inhibited in the absence of CD44. Treatment with a blocking antibody to CD44 reduced lung fibrosis in mice in vivo. Finally, fibroblasts isolated from patients with IPF exhibited an invasive phenotype that was also dependent on HAS2 and CD44. Understanding the mechanisms leading to an invasive fibroblast phenotype could lead to novel approaches to the treatment of disorders characterized by severe tissue fibrosis.

Lymphocyte apoptosis is resistant to erythropoietin in porcine endotoxemia.

Sepsis-induced lymphocyte apoptosis plays an important role in the development of immune suppression in septic patients. Erythropoietin (EPO) is a multifunctional cytokine with antiapoptotic properties. We hypothesized that EPO could mitigate mononuclear cell (MNC) apoptosis and modify the dynamic changes of MNCs during endotoxemia. Twenty-six pigs were randomized into three groups: (i) lipopolysaccharides (LPS), (ii) EPO (epoetin-α, 5000 IU/kg) administered 60 min prior to LPS, and (iii) sham. At 120 min of endotoxemia, the animals were fluid resuscitated and the LPS infusion was reduced. MNCs were isolated at 0, 60, 240, and 540 min of endotoxemia, and apoptosis was assessed by flow cytometry. Apoptosis in splenic biopsies was quantified by immunohistochemistry. Endotoxemia increased the number of apoptotic MNCs in the blood (p ≤ 0.01) and the spleen (p = 0.03), and EPO did not modify this increase. The number of T-helper and cytotoxic T cells declined during endotoxemia. The dynamic changes of the MNC subsets were not modified by treatment with EPO. In conclusion, EPO did not modify the LPS-induced changes of MNC subsets or mitigate the levels of apoptosis of MNCs in the blood or in the spleen. This study does not support that EPO confers protection against lymphocyte apoptosis.