PreImplantation Factor bolsters neuroprotection via modulating Protein Kinase A and Protein Kinase C signaling.

A synthetic peptide (sPIF) analogous to the mammalian embryo-derived PreImplantation Factor (PIF) enables neuroprotection in rodent models of experimental autoimmune encephalomyelitis and perinatal brain injury. The protective effects have been attributed, in part, to sPIF's ability to inhibit the biogenesis of microRNA let-7, which is released from injured cells during central nervous system (CNS) damage and induces neuronal death. Here, we uncover another novel mechanism of sPIF-mediated neuroprotection. Using a clinically relevant rat newborn brain injury model, we demonstrate that sPIF, when subcutaneously administrated, is able to reduce cell death, reverse neuronal loss and restore proper cortical architecture. We show, both in vivo and in vitro, that sPIF activates cyclic AMP dependent protein kinase (PKA) and calcium-dependent protein kinase (PKC) signaling, leading to increased phosphorylation of major neuroprotective substrates GAP-43, BAD and CREB. Phosphorylated CREB in turn facilitates expression of Gap43, Bdnf and Bcl2 known to have important roles in regulating neuronal growth, survival and remodeling. As is the case in sPIF-mediated let-7 repression, we provide evidence that sPIF-mediated PKA/PKC activation is dependent on TLR4 expression. Thus, we propose that sPIF imparts neuroprotection via multiple mechanisms at multiple levels downstream of TLR4. Given the recent FDA fast-track approval of sPIF for clinical trials, its potential clinical application for treating other CNS diseases can be envisioned.

Heat shock protein upregulation lowers cytokine levels after ischemia and reperfusion.

OBJECTIVE: Studies showed that the expression of heat shock protein 70 (HSP70) by whole-body hyperthermia or warming of the heart is associated with protection against ischemia/reperfusion injury. The aim of this study is to determine a time-related response of HSP70 expression through topical cardiac warming with correlation to cytokine production. METHODS: 30 rats were divided into three groups: no heat shock, heat shocked, and controls. Heat shock was performed with 42 degrees C saline solution applied to the heart for 5, 30, and 60 min. HSP70 and cytokines were measured. RESULTS: Heat shock treated animals showed a 1.2-fold increase after 5 min (NS) in HSP70 expression, a 2.0-fold increase (p < 0.02) after 30 min, and a 2.3-fold increase (p < 0.012) after 60 min compared to controls. The IL-1beta levels decreased from 14.3 pg/ml (normal controls) to 7.1 pg/ml after 5 min, to 1.6 pg/ml after 30 min (p < 0.002), and to 1.4 pg/ ml after 60 min of heat shock treatment (p < 0.002). The TNF-alpha levels also decreased, but not significantly. CONCLUSIONS: Upregulation of HSP70 through this novel method is instant and detectable within hours. The amount of HSP70 expression induced is time dependent, showing an indirect correlation with cytokine levels. These results suggest that the protective effect of HSP70 is immediate and might be explained by reduced cytokine levels. No prior recovery period is needed.

Scaffold precoating with human autologous extracellular matrix for improved cell attachment in cardiovascular tissue engineering.

Cell attachment to a scaffold is a precondition for the development of bioengineered valves and vascular substitutes. This attachment is generally facilitated by the use of precoating factors, but some can cause toxic or immunologic side effects. Autologous extracellular matrix (ECM) is used as a precoating factor in our study. Ascending aortic tissue was cultured to obtain human myofibroblasts. Autologous ECM was extracted from the same aortic tissue. Poly(glycolic acid) (PGA) scaffolds were precoated with autologous ECM, human serum, or poly-L-lysine; the control group was pretreated with phosphate buffered saline (PBS). Myofibroblasts were seeded onto each scaffold, and the cell attachment was assayed and compared. Compared with the control group, precoating with human serum, poly-L-lysine, and ECM increased number of attached cells by 24%, 53%, and 48%, respectively. Differences between precoating groups were significant (p < 0.01), except for ECM versus poly-L-lysine. Scanning electron microscopy also demonstrated the high degree of cell attachment to the PGA fibers on scaffolds precoated with ECM and poly-L-lysine. Precoating polymeric scaffold with autologous human extracellular matrix is a very effective method of improving cell attachment in cardiovascular tissue engineering without the potential risk of immunologic reactions.

Expression of adhesion molecules and cytokines after coronary artery bypass grafting during normothermic and hypothermic cardiac arrest.

OBJECTIVE: Cardiac surgery with cardiopulmonary bypass (CPB) results in vascular injury and tissue damage which involves leukocyte-endothelial interactions mediated by cytokines and adhesion molecules. This study was designed to demonstrate the effect of normothermic and hypothermic CPB to cytokine and soluble adhesion molecule levels in adults and to determine whether these levels correlate to the patients postoperative course. DESIGN AND PATIENTS: In 25 patients after normothermic and in 25 patients after hypothermic coronary artery bypass grafting with cardiopulmonary bypass (CPB), blood samples for cytokine and soluble adhesion molecule analysis were taken preoperatively, 24, 36, 48 h, and 6 days postoperatively. Soluble adhesion molecules (sE-selectin, sICAM-1) were measured by ELISA and cytokines (TNF-alpha, IL-6, IL-8) by chemilumenscent-immunoassay. Clinical data were collected prospectively. RESULTS: Postoperatively, adhesion molecule and cytokine levels were significantly elevated after CPB. Mean plasma levels of sICAM-1 was 2.4-fold higher after 6 days. Mean plasma concentration of sE-selectin peaked after 48 h with a 2-fold increase compared to normothermic conditions. In the hypothermia group sICAM-1, sE-selectin, IL-6, and IL-8 showed significantly higher levels (P<0.0057, P<0.0012, P<0.0419, P<0.0145) after 24 h compared to the normothermia group. No clinical differences were seen. CONCLUSION: Adhesion molecules and cytokines are elevated after CPB. Patients after hypothermic CPB show significant higher sICAM-1, sE-selectin, IL-6, and IL-8 levels after 24 h compared to normothermic conditions. These results are mainly due to longer CPB and crossclamp times but do not alter the patient's postoperative course.

Tissue engineering in cardiovascular surgery: new approach to develop completely human autologous tissue.

OBJECTIVE: In cardiovascular tissue engineering, three-dimensional scaffolds serve as physical supports and templates for cell attachment and tissue development. Currently used scaffolds are still far from ideal, they are potentially immunogenic and they show toxic degradation and inflammatory reactions. The aim of this study is to develop a new method for a three-dimensional completely autologous human tissue without using any scaffold materials. METHODS: Human aortic tissue is harvested from the ascending aorta in the operation room and worked up to pure human myofibroblasts cultures. These human aortic myofibroblasts cultures (1.5x10(6) cells, passage 3) were seeded into 15-cm culture dishes. Cells were cultured with Dulbecco' s modified Eagle's medium supplemented with 1 mM L-ascorbic acid 2-phosphate for 4 weeks to form myofibroblast sheets. The harvested cell sheets were folded to form four-layer sheets. The folded sheets were then framed up and cultured for another 4 weeks. Tissue development was evaluated by biochemical assay and light and electron microscopy. RESULTS: After 4 weeks of culture in ascorbic acid supplemented medium, myofibroblasts formed thin cell sheets in culture dishes. The cell sheets presented in a multi-layered pattern surrounded by extracellular matrices. Cultured for additional 4 weeks on the frames, the folded sheets further developed into more solid and flexible tissues. Light microscopy documented a structure resembling to a native tissue with confluent extracellular matrix. Under transmission electron microscope, viable cells and confluent bundles of striated mature collagen fibers were observed. Hydroxyproline assays showed significant increase of collagen content after culturing on the frames and were 80.5% of that of natural human pericardium. CONCLUSIONS: Improved cell culture technique may render human aortic myofibroblasts to a native tissue-like structure. A three-dimensional completely autologous human tissue may be further developed on the base of this structure with no show toxic degradation or inflammatory reactions.

Modified ultrafiltration lowers adhesion molecule and cytokine levels after cardiopulmonary bypass without clinical relevance in adults.

OBJECTIVE: Cardiac surgery with cardiopulmonary bypass (CPB) results in expression of cytokines and adhesion molecules (AM) with subsequent inflammatory response. The purpose of the study was to evaluate the clinical impact of modified ultrafiltration (MUF) and its efficacy in reducing cytokines and AM following coronary artery bypass grafting (CABG) in adults. METHODS: A prospective randomized study of 97 patients undergoing elective CABG was designed. Fifty patients were operated on using normothermic and 47 patients using hypothermic CPB. The normothermic group was subdivided into a group with modified ultrafiltration (n = 30) and a group without MUF (n = 20). In the hypothermic group 30 patients received MUF compared to 17 patients serving as controls. MUF was instituted after CPB for 15 min through the arterial and venous bypass circuit lines. Cytokines (IL-6, IL-8, TNF-alpha, IL-2R) and adhesion molecules (sE-selectin, sICAM-1) were measured preoperatively, pre-MUF, in the ultrafiltrate, 24 h, 48 h and 6 days after surgery by chemiluminescent enzyme immunometric assay or enzyme-linked immunosorbent assay (ELISA). Clinical parameters were collected prospectively until discharge. RESULTS: In all patients AM and cytokines were significantly elevated after normothermic and hypothemic CPB. AM and cytokines were significantly higher in hypothermia compared to normothermia. In hypothermic CPB sE-selectin was decreased after 24 h by 37% (P < 0.0063) and by 40% (P < 0.0027) after 48 h postoperatively. ICAM-1 was reduced by 43% (P < 0.0001) after 24 h and by 60% (P < 0.0001) after 6 days. Similar results were seen in cytokines with reduction up to 60% after 24 h. Changes after 48 h were noticeable but not significant. Reduction of AM and cytokines after normothermic CPB was minimal. Neither in normothermia, nor in hypothermia has sIL-2R been effectively removed from the circulation. There were no significant differences in the clinical variables between the patients with or without MUF. CONCLUSION: AM and cytokines are significantly elevated after hypothermic CPB compared to normothermic CPB. MUF led to a significant reduction in cytokine and AM levels after hypothermic CPB, except for IL-2R. MUF showed minimal effect in normothermia. We conclude that MUF is an efficient way to remove cytokines and AM. However, we were unable to demonstrate any significant impact of MUF in outcome of adults after elective CABG.

Optimized growth conditions for tissue engineering of human cardiovascular structures.

Optimized in vitro formation of strong tissue is a prerequisite for tissue engineering of cardiovascular structures, such as heart valves and blood vessels. This study evaluates different growth media additives as to cell proliferation, extracellular matrix formation, and mechanical characteristics. Biodegradable polymers were seeded with human vascular myofibroblasts. Group A was cultured with standard medium, groups B, C, and D were in addition supplemented with ascorbate, fibroblast growth factor (bFGF), and both respectively. Analysis included histology, electron microsocopy, mechanical testing, and biochemical assays for cell proliferation (DNA) and extracellular matrix (collagen). DNA content increased in all groups, showing significantly more cells in group C and D after 14d. Collagen increased in all groups, except for C. Morphology showed viable, layered cellular tissue, with collagen fibrils after 2w, most pronounced in B and D. Mechanical properties decreased initially, stabilizing after 2w. In conclusion, standard nutrient media were efficient for seeded human vascular cells cultured on biodegradable meshes. Supplementation with bFGF+ascorbate resulted in enhanced early cell proliferation and structurally more mature tissue formation.

Tissue engineering of a bioprosthetic heart valve: stimulation of extracellular matrix assessed by hydroxyproline assay.

Creation of an autologous heart valve by tissue engineering offers a promising approach to cardiac surgery. Although we have demonstrated successful formation of native valve analogous tissue in vitro, hemodynamic competence remains a serious problem. The aim of this study was to optimize in vitro formation of collagen as a precondition for mechanical stability of new tissue. Human myofibroblasts were seeded on square sheets of biodegradable scaffolds (control). To stimulate collagen production, one series was cultured with L-ascorbic acid 2-phosphate. In a second series, the seeded scaffolds were subjected to tension by mounting them on a frame. After 4 weeks of culture time, the collagen content of the different series was assessed by hydroxyproline assay. Light and scanning electron microscopy were performed. Hydroxyproline content of the framed scaffolds was 10 times higher than that of the control group (p < 0.05) and 6 times higher than in the unframed scaffolds grown with ascorbic acid (p < 0.05), respectively. Scanning electron microscopy proved extensive formation of solid tissue in the framed samples. These results demonstrate that supplementation of myofibroblast cultures with ascorbic acid, especially if grown on strained scaffolds, significantly increases collagen content, which is crucial for mechanical stability. This concept is a further step toward the creation of a hemodynamically competent autologous heart valve.

Tissue engineering in cardiovascular surgery: MTT, a rapid and reliable quantitative method to assess the optimal human cell seeding on polymeric meshes.

OBJECTIVE: Currently used valve substitutes for valve replacement have certain disadvantages that limit their long-term benefits such as poor durability, risks of infection, thromboembolism or rejection. A tissue engineered autologous valve composed of living tissue is expected to overcome these shortcomings with natural existing biological mechanisms for growth, repair, remodeling and development. The aim of the study was to improve cell seeding methods for developing tissue-engineered valve tissue. METHODS: Human aortic myofibroblasts were seeded on polyglycolic acid (PGA) meshes. Cell attachment and growth of myofibroblasts on the PGA scaffolds with different seeding intervals were compared to determine an optimal seeding interval. In addition, scanning electron microscopy study of the seeded meshes was also performed to document tissue development. RESULTS: There was a direct correlation between cell numbers assessed by direct counting and MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltertra-zoliu m bromide) assay. Both attach rate and cell growth seeded on meshes with long intervals (24 and 36 h) were significantly higher than those seeded with short intervals (2 and 12 h) (P<0.01), there was no significant difference between 24- and 36-h seeding interval. Scanning electron microscopy also documented more cell attachment with long seeding intervals resulting in a more solid tissue like structure. CONCLUSION: It is feasible to use human aortic myofibroblasts to develop a new functional tissue in vitro. Twenty-four hours is an optimal seeding interval for seeding human aortic myofibroblasts on PGA scaffolds and MTT test is a rapid and reliable quantitative method to assess the optimal human cell seeding on polymeric meshes.

Tissue engineering: a new approach in cardiovascular surgery--seeding of human fibroblasts on resorbable mesh.

INTRODUCTION: In tissue engineering the material properties of synthetic compounds are manipulated to enable delivery of dissociated cells onto a scaffold in a manner that will result in in vitro formation of new functional tissue. The seeding of human fibroblasts on resorbable mesh is a precondition of a successful creation of human tissue such as autologous cardiac valves. MATERIAL AND METHODS: Polymeric scaffolds (n = 12) composed of polyglycolic acid (PGA) with a fiber diameter of 12-15 mm and a polymer density of 70 mg/ml were used as square sheets of 0.3 x 1 x 1 cm. Fibroblasts (passage 5), harvested from human foreskin, were seeded (3.4 x 10(6)) and cultured over a 3 week period on a PGA mesh. RESULTS: Microscopic examination of the seeded mesh demonstrated that the human fibroblasts were attached to the polymeric fibers and had begun to spread out and to divide. Electron microscopy showed a continuous distribution and formation of the cells throughout the "polymeric architecture". Spotlike hydrolysis of PGA fibers was observed. After 3 weeks the seeded scaffolds resembled a solid sheet of tissue. CONCLUSION: These preliminary results, successful seeding of human fibroblasts on a PGA mesh, represent a first basic step on the way to construct human tissue such as autologous cardiac valves and demonstrate that tissue engineering might be a promising new device in therapy of cardiovascular disease.

[The significance of endothelial adhesion molecules in heart surgery]. / Die Bedeutung der endothelialen Adhäsionsmoleküle in der Herzchirurgie.

Myocardial ischemia and reperfusion is a common event in cardiovascular surgery patients. The endothelium has been shown to play a key role in the injury suffered after ischemia and reperfusion. When endothelial cells became hypoxic followed by reoxygenation they become activated to express endothelial adhesion molecules followed by the migration of neutrophils into the tissue. These changes may contribute to the early postoperatively myocardial dysfunction. An increased understanding of the interaction between leukocytes and endothelium may allow to develop new therapies in future.

Tissue engineering: a new approach in cardiovascular surgery: Seeding of human fibroblasts followed by human endothelial cells on resorbable mesh.

OBJECTIVE: In tissue engineering the material properties of synthetic compounds are chosen to enable delivery of dissociated cells onto a scaffold in a manner that will result in in vitro formation of a new functional tissue. The seeding of human fibroblasts followed by human endothelial cells on resorbable mesh is a precondition of a successful creation of human tissues such as vessels or cardiac valves. METHODS: Polymeric scaffolds (n = 18) composed of polyglycolic acid (PGA) with a fiber diameter of 12-15 microm and a polymer density of 70 mg/ml were used as square sheets of 1 x 1 x 0.3 cm. Fibroblasts (passage 7) harvested from human foreskin were seeded (3.4 x 10(6)) and cultured over a 3 week period on a PGA-mesh, followed by seeding of endothelial cells (passage 5, 2.8 x 10(6)) harvested from human ascending aorta. Thereafter the new tissue was stained for HE, van Gieson, Trichrom Masson, Factor VIII and CD 34 and proved by scanning electron microscopy. RESULTS: Microscopic examination of the seeded mesh demonstrated that the human fibroblasts were attached to the polymeric fibers and had begun to spread out and divide. The scanning electron microscopic examination demonstrated a homogeneous scaffold resembling a solid sheet of tissue. The seeded endothelial cells formed a monolayer on the fibroblasts and no endothelial cell invasion or new formation of capillaris could be detected. CONCLUSIONS: These results are a first step to demonstrate that seeding of human fibroblasts and endothelial cells on PGA-mesh might be a feasible model to construct human tissues such as vessels or cardiac valves.

Fluorescence activated cell sorting: a reliable method in tissue engineering of a bioprosthetic heart valve.

BACKGROUND: Techniques of tissue engineering are used to seed human autologous cells in vitro on degradable mesh to create new functional tissue like a bioprosthetic heart valve. A precondition is subsequent seeding of native-valve-analogous pure endothelial and myofibroblast cell lines. The aim of this study is to find a safe method of isolating viable cell lines out of tissues from the operating room. METHODS: Mixed cells from ascending aorta obtained from the operating room were incubated with an endothelial-specific fluorescent marker. The labeled cells were activated and sorted by flow cytometry. Isolated cell lines were cultured and thereafter square sheets of polymeric scaffold were seeded with myofibroblasts, followed by endothelial cells. The created tissue was stained with hematoxylin and eosin, van Gieson stain, and stains for factor VIII and CD34. RESULTS: Control culture samples (n = 25) revealed vital uncontaminated endothelial and myofibroblast cell lines. Microscopy of the seeded meshes (n = 16) demonstrated a tissue-like structure. Van Gieson stain showed production of collagen. Endothelial cells formed a superficial monolayer, demonstrated by factor VIII and CD34; no invasive formation of capillaries was detectable. CONCLUSIONS: These results demonstrate that fluorescence activated cell sorting is a reliable and safe method to gain pure vital autologous cell lines out of human mixed cells for subsequent seeding on degradable mesh and that those cells are active to form new tissue.

A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle.

An exceptional muscle development commonly referred to as 'double-muscled' (Fig. 1) has been seen in several cattle breeds and has attracted considerable attention from beef producers. Double-muscled animals are characterized by an increase in muscle mass of about 20%, due to general skeletal-muscle hyperplasia-that is, an increase in the number of muscle fibers rather than in their individual diameter. Although the hereditary nature of the double-muscled condition was recognized early on, the precise mode of inheritance has remained controversial; monogenic (domainant and recessive), oligogenic and polygenic models have been proposed. In the Belgian Blue cattle breed (BBCB), segregation analysis performed both in experimental crosses and in the outbred population suggested an autosomal recessive inheritance. This was confirmed when the muscular hypertrophy (mh) locus was mapped 3.1 cM from microsatellite TGLA44 on the centromeric end of bovine chromosome 2 (ref. 5). We used a positional candidate approach to demonstrate that a mutation in bovine MSTN, which encodes myostatin, a member of the TGF beta superfamily, is responsible for the double-muscled phenotype. We report an 11-bp deletion in the coding sequence for the bioactive carboxy-terminal domain of the protein causing the muscular hypertrophy observed in Belgian Blue cattle.

Characterization of a set of variable number of tandem repeat markers conserved in bovidae.

Screening purpose-built libraries with minisatellite probes, we have isolated 36 bovine variable number of tandem repeat markers (VNTRs) characterized by a mean heterozygosity of 59.3 within the American Holstein breed. Matching probabilities and exclusion powers were estimated by Monte-Carlo simulation, showing that the top 5 to 10 markers could be used as a very efficient DNA-based system for individual identification and paternity diagnosis. The isolated VNTR systems should contribute significantly to the establishment of a bovine primary DNA marker map. Linkage analysis, use of somatic cell hybrids, and in situ hybridization demonstrate that these bovine VNTRs are scattered throughout the bovine genome, without evidence for proterminal confinement as in the human, and that at least some of them are organized as clusters. Moreover, Southern blot analysis and in situ hybridization demonstrate conservation of sequence and map location of minisatellites within Bovidae.