Whole-body protochordate regeneration from totipotent blood cells.

Cell differentiation, tissue formation, and organogenesis are fundamental patterns during the development of multicellular animals from the dividing cells of fertilized eggs. Hence, the complete morphogenesis of any developing organism of the animal kingdom is based on a complex series of interactions that is always associated with the development of a blastula, a one-layered hollow sphere. Here we document an alternative pathway of differentiation, organogenesis, and morphogenesis occurring in an adult protochordate colonial organism. In this system, any minute fragment of peripheral blood vessel containing a limited number of blood cells isolated from Botrylloides, a colonial sea squirt, has the potential to give rise to a fully functional organism possessing all three embryonic layers. Regeneration probably results from a small number of totipotent stem cells circulating in the blood system. The developmental process starts from disorganized, chaotic masses of blood cells. At first an opaque cell mass is formed. Through intensive cell divisions, a hollow, blastula-like structure results, which may produce a whole organism within a short period of a week. This regenerative power of the protochordates may be compared with some of the characteristics associated with the formation of mammalian embryonal carcinomous bodies. It may also serve as an in vivo model system for studying morphogenesis and differentiation by shedding more light on the controversy of the "stem cell" vs. the "dedifferentiation" theories of regeneration and pattern formation.

Spatial correlations between beta-amyloid (Abeta) deposits and blood vessels in early-onset Alzheimer's disease

In cases of late-onset Alzheimer’s disease (AD), there is a spatial correlation between the classsic ‘cored’ type of Beta-amyloid (Abeta) deposit and the large vertically penetrating arterioles in the cerebral cortex suggesting that blood vessels are involved in the pathogenesis of the classic deposits. In this chapter, the spatial correlations between the diffuse, primitive, and classic Abeta deposits and blood vessels were studied in 10 cases of early-onset AD in the age range 40 – 65 years. Sections of frontal cortex were immunostained with antibodies against Abeta?and with collagen IV to reveal the Abeta deposits and blood vessel profiles. In the early-onset cases as a whole, all types of Abeta? deposit and blood vessel profiles were distributed in clusters. There was a positive spatial correlation between the clusters of the diffuse Abeta deposits and the larger (>10µm) and smaller diameter (<10?m) blood vessel profiles in one and three cases respectively. The primitive and classic Abeta deposits were spatially correlated with larger and smaller blood vessels both in three and four cases respectively. Spatial correlations between the Abeta deposits and blood vessels may be more prevalent in cases expressing amyloid precursor protein (APP) than presenilin 1 (PSEN1) mutations. Apolipoprotein E (Apo E) genotype of the patient did not appear to influence the spatial correlation with blood vessel profiles. The data suggest that the larger diameter blood vessels are less important in the pathogenesis of the classic Abeta deposits in early-onset compared with late-onset AD.

Spatial correlations between beta-amyloid (A beta) deposits and blood vessels in familial Alzheimer's disease

In sporadic Alzheimer’s disease (SAD), the classic (‘dense-cored’) ß-amyloid (Aß) deposits are aggregated around the larger blood vessels in the upper laminae of the cerebral cortex. To determine whether a similar relationship exists in familial AD (FAD), the spatial correlations between the diffuse, primitive, and classic ß-amyloid (Aß deposits and blood vessels were studied in ten FAD cases including cases linked to amyloid precursor protein (APP) and presenilin (PSEN) gene mutations and expressing apolipoprotein E (apo E) allele E4. Sections of frontal cortex were immunolabelled with antibodies against Aß and with collagen IV to reveal the Aß deposits and blood vessel profiles. In the FAD cases as a whole, Aßdeposits were distributed in clusters. There was a positive spatial correlation between the clusters of the diffuse Aßdeposits and the larger (>10 µm) and smaller diameter (<10 µm) blood vessels in one and three cases respectively. The primitive Aß deposits were spatially correlated with larger and smaller blood vessels each in four cases and the classic deposits in three and four cases respectively. Apo E genotype of the patient did not influence spatial correlation with blood vessels. Hence, spatial correlations between the classic deposits and larger diameter blood vessels were significantly less frequent in FAD compared with SAD. It was concluded that both Aß deposit morphology and AD subtype determine spatial correlations with blood vessels in AD.

Classic beta-amyloid deposits cluster around large diameter blood vessels rather than capillaries in sporadic Alzheimer's disease

Various hypotheses could explain the relationship between beta-amyloid (Abeta) deposition and the vasculature in Alzheimer's disease (AD). Amyloid deposition may reduce capillary density, affect endothelial cells of blood vessels, result in diffusion from blood vessels, or interfere with the perivascular clearance mechanism. Hence, the spatial pattern of the classic ('cored') type of Abeta deposit was studied in the upper laminae (I,II/III) of the superior frontal gyrus in nine cases of sporadic AD (SAD). Sections were immunostained with antibodies against Abeta and with collagen IV to study the relationships between the spatial distribution of the classic deposits and the blood vessel profiles. Both the classic deposits and blood vessel profiles were distributed in clusters. In all cases, there was a positive spatial correlation between the clusters of the classic deposits and the larger diameter (>10 microm) blood vessel profiles and especially the vertically penetrating arterioles. In only 1 case, was there a significant spatial correlation between the clusters of the classic deposits and the smaller diameter (<10 microm) capillaries. There were no negative correlations between the density of Abeta deposits and the smaller diameter capillaries. In 9/11 cases, the clusters of the classic deposits were significantly larger than those of the clusters of the larger blood vessel profiles. In addition, the density of the classic deposits declined as a negative exponential function with distance from a vertically penetrating arteriole. These results suggest that the classic Abeta deposits cluster around the larger blood vessels in the upper laminae of the frontal cortex. This aggregation could result from diffusion of proteins from blood vessels or from overloading the system of perivascular clearance from the brain.

Florid prion protein (PrP) plaques in patients with variant Creutzfeldt-Jakob disease (vCJD) are spatially related to blood vessels

This study tested the hypothesis that variations in the density of the florid prion protein (PrP) plaques in the brain of patients with variant Creutzfeldt-Jakob disease (vCJD) were spatially related to blood vessels. In 81% of areas of the cerebral cortex sampled and in 37% of the remaining areas, which included the hippocampus, dentate gyrus, and cerebellum, there was a positive spatial correlation between the density of the florid plaques and the larger blood vessel profiles. The frequency of the positive spatial correlations was similar in different anatomical areas of the cerebral cortex and in the upper compared with the lower cortical laminae. The data support the hypothesis that the florid plaques cluster around the larger blood vessels in vCJD, the density of associated plaques increasing with vessel size. The development of florid plaques close to blood vessels may be due to factors associated with the blood vessels that enhance the aggregation of PrP to form the dense cores of florid plaques and is unlikely to reflect the haematogenous spread of PrP into the brain.

Pathological changes in the sporadic and variant forms Of Creutzfeldt-Jakob Disease (CJD) are spatially related to blood vessels

The objective of this article was to determine whether the pathological changes of Creutzfeldt-Jacob disease (CJD) were related to the brain microcirculation. Hence, the spatial correlations between the vacuolation, prion protein (PrP) deposits, and the blood vessel profiles were studied in immunolabelled sections of the cerebral cortex, hippocampus, and cerebellum in two subtypes of CJD, viz., sporadic CJD (sCJD) and variant CJD (vCJD). In sCJD, both the vacuolation and the ‘synaptic-type’ PrP deposits were spatially correlated with the microvessels; the PrP deposits being more strongly correlated than the vacuoles. In vCJD, there were no significant spatial correlations between either the vacuolation or the diffuse-type of PrP deposit and the microvessels. By contrast, a consistent pattern of spatial correlation was observed in gyri of the cerebral cortex between the florid PrP deposits and microvessels. In both sCJD and vCJD, the frequency of positive spatial correlations was similar in the different gyri of the cerebral cortex and in the upper compared with the lower laminae. In conclusion, the microcirculation may be more significantly involved in determining the pathological changes in sCJD than in vCJD. The spatial correlations of the florid PrP deposits in vCJD and the synaptic deposits in sCJD and the blood vessels may be attributable to factors associated with the microcirculation which enhance the aggregation of PrP molecules rather than representing a possible haematogenous spread of the disease. S

Dispersion of classic beta-amyloid deposits around blood vessels in Alzheimer’s disease

The spatial pattern of the classic (‘cored’) type of beta-amyloid (Abeta) deposit was studied in the upper laminae of the superior temporal gyrus in 9 cases of sporadic Alzheimer’s disease (SAD). Abeta stained tissue was counterstained with collagen IV to study the relationships between the spatial distribution of the classic deposits and the blood vessel profiles. Both the classic deposits and blood vessel profiles were distributed in clusters. In all cases, there was a spatial correlation between the clusters of the classic deposits and the larger diameter (>10 micron) blood vessel profiles and especially the vertically penetrating arterioles. In only 1 case, was there a significant spatial correlation between the clusters of the classic deposits and the smaller diameter (<10 micron) capillaries. In 9/11 cases, the clusters of the classic deposits were significantly larger than those of the clusters of the larger blood vessels. In addition, the density of the classic deposits declined as a negative exponential function with distance from the vertically penetrating arterioles. These results suggest that the classic Abeta deposits cluster around the larger blood vessels in the frontal cortex and that diffusion of proteins from these blood vessels could be involved in the pathogenesis of the classic deposits in SAD.

Relationships between beta-amyloid (Abeta) deposits and blood vessels in familial and sporadic Alzheimer's disease

Introduction: The density of diffuse, primitive and classic beta-amyloid (Abeta) deposits and blood vessels was studied in nine cases of sporadic Alzheimer's disease (SAD) and 10 cases of familial Alzheimer's disease (FAD) including two cases with amyloid precursor protein (APP) mutations (APP717, Val - Ile). Materials and Methods: Sections of frontal cortex stained for Abeta12-28 counterstained with collagen type IV antiserum. Densities measured along the upper cortex in 64-128, 1000 x 200 micron continuous sample fields. Results: The density of diffuse and primitive deposits was not correlated with blood vessels in FAD or SAD. The density of the classic deposits was positively correlated with the larger diameter (> 10 micron) blood vessels in all SAD cases and weakly correlated with blood vessel in three non-APP FAD cases. Conclusions: Blood vessels are less important in the formation of classic Abeta deposits in FAD compared with SAD.

Dispersion of prion protein deposits around blood vessels in variant Creutzfeldt-Jakob disease

In variant Creutzfeldt-Jakob disease (vCJD), a disease linked to bovine spongiform encephalopathy (BSE), florid-type prion protein (PrP(sc)) deposits are aggregated around the larger diameter (> 10 µm) cerebral microvessels. Clustering of PrP(sc) deposits around blood vessels may result from blood-borne prions or be a consequence of the cerebral vasculature influencing the development of the florid deposits. To clarify the factors involved, the dispersion of the florid PrP(sc) deposits was studied around the larger diameter microvessels in the neocortex, hippocampus, and cerebellum of ten cases of vCJD. In the majority of brain regions, florid deposits were clustered around the larger diameter vessels with a mean cluster size of between 50 µm and 628 µm. With the exception of the molecular layer of the dentate gyrus, the density of the florid deposits declined as a negative exponential function of distance from a blood vessel profile suggesting that diffusion of molecules from blood vessels is a factor in the formation of the florid deposits. Diffusion of PrP(sc) directly into the brain via the microvasculature has been demonstrated in vCJD in a small number of cases. However, the distribution of the prion deposits in vCJD is more likely to reflect molecular 'chaperones' diffusing from vessels and promoting the aggregation of pre-existing PrP(sc) in the vicinity of the vessels to form florid deposits.

Pathological changes in the sporadic and variant forms of Creutzfeldt-Jakob Disease (CJD) are spatially related to blood vessels

The objective of this article was to determine whether the pathological changes of Creutzfeldt-Jacob disease (CJD) were related to the brain microcirculation. Hence, the spatial correlations between the vacuolation, prion protein (PrP) deposits, and the blood vessel profiles were studied in immunolabelled sections of the cerebral cortex, hippocampus, and cerebellum in two subtypes of CJD, viz., sporadic CJD (sCJD) and variant CJD (vCJD). In sCJD, both the vacuolation and the ‘synaptic-type’ PrP deposits were spatially correlated with the microvessels; the PrP deposits being more strongly correlated than the vacuoles. In vCJD, there were no significant spatial correlations between either the vacuolation or the diffuse-type of PrP deposit and the microvessels. By contrast, a consistent pattern of spatial correlation was observed in gyri of the cerebral cortex between the florid PrP deposits and microvessels. In both sCJD and vCJD, the frequency of positive spatial correlations was similar in the different gyri of the cerebral cortex and in the upper compared with the lower laminae. In conclusion, the microcirculation may be more significantly involved in determining the pathological changes in sCJD than in vCJD. The spatial correlations of the florid PrP deposits in vCJD and the synaptic deposits in sCJD and the blood vessels may be attributable to factors associated with the microcirculation which enhance the aggregation of PrP molecules rather than representing a possible haematogenous spread of the disease.

Induction of placental heme oxygenase-1 is protective against TNFα-induced cytotoxicity and promotes vessel relaxation

Background: Pregnancy is characterized by an inflammatory-like process and this may be exacerbated in preeclampsia. The heme oxygenase (HO) enzymes generate carbon monoxide (CO) that induces blood vessel relaxation and biliverdin that acts as an endogenous antioxidant. Materials and Methods: We examined the expression and localization of HO-1 and HO-2 in normal and preeclamptic placenta using reverse transcription polymerase chain reaction (RT-PCR), RNase protection assay, immunoblotting and immunohistochemistry. In addition, the effect of HO activation on tumor necrosis factor-alpha (TNF) induced placental damage and on feto-placental circulation was studied. Results: We provide the first evidence for the role of HO as an endogenous placental factor involved with cytoprotection and placental blood vessel relaxation. HO-1 was significantly higher at term, compared with first trimester placentae indicating its role in placental vascular development and regulation. HO-1 predominantly localized in the extravascular connective tissue that forms the perivascular contractile sheath around the developing blood vessels. HO-2 was localized in the capillaries, as well as the villous stroma, with weak staining of trophoblast. Induction of HO-1 caused a significant attenuation of TNF-mediated cellular damage in placental villous explants, as assessed by lactate dehydrogenase leakage (p 0.01). HO-1 protein was significantly reduced in placentae from pregnancies complicated with preeclampsia, compared with gestationally matched normal pregnancies. This suggests that the impairment of HO-1 activation may compromise the compensatory mechanism and predispose the placenta to cellular injury and subsequent maternal endothelial cell activation. Isometric contractility studies showed that hemin reduced vascular tension by 61% in U46619-preconstricted placental arteries. Hemininduced vessel relaxation and CO production was inhibited by HO inhibitor, tin protoporphyrin IX. Conclusions: Our findings establish HO-1 as an endogenous system that offers protection against cytotoxic damage in the placenta, identifies the HO-CO pathway to regulate feto-placental circulation and provides a new approach to study the disease of preeclampsia.

Scaffold-free, Human Mesenchymal Stem Cell-Based Tissue Engineered Blood Vessels.

Tissue-engineered blood vessels (TEBV) can serve as vascular grafts and may also play an important role in the development of organs-on-a-chip. Most TEBV construction involves scaffolding with biomaterials such as collagen gel or electrospun fibrous mesh. Hypothesizing that a scaffold-free TEBV may be advantageous, we constructed a tubular structure (1 mm i.d.) from aligned human mesenchymal cell sheets (hMSC) as the wall and human endothelial progenitor cell (hEPC) coating as the lumen. The burst pressure of the scaffold-free TEBV was above 200 mmHg after three weeks of sequential culture in a rotating wall bioreactor and perfusion at 6.8 dynes/cm(2). The interwoven organization of the cell layers and extensive extracellular matrix (ECM) formation of the hMSC-based TEBV resembled that of native blood vessels. The TEBV exhibited flow-mediated vasodilation, vasoconstriction after exposure to 1 μM phenylephrine and released nitric oxide in a manner similar to that of porcine femoral vein. HL-60 cells attached to the TEBV lumen after TNF-α activation to suggest a functional endothelium. This study demonstrates the potential of a hEPC endothelialized hMSC-based TEBV for drug screening.

Profile of cortisol, glycaemia, and blood parameters of American Bullfrog tadpoles Lithobates catesbeianus exposed to density and hypoxia stressors

O objetivo deste estudo foi avaliar a resposta fisiológica (cortisol, glicemia e parâmetros sanguineos) de girinos de rã-touro (Lithobates catesbeianus) em diferentes densidades e após exposição aérea. Os animais utilizados no experimento estavam entre os estágios 31 a 39, na fase de pró-metamorfose sendo testados 1 girino/L (Tratamento 1), 5 girinos/L (Tratamento 2) e 10 girinos/L (Tratamento 3), conduzidos em 3 réplicas simultâneas durante 12 dias. O sangue foi retirado por rompimento do vaso caudal na condição de Normóxia - N (tempo zero) e Hipóxia - H (tempo de 15 minutos de exposição ao ar). Foi observado um aumento nos valores de cortisol, aos 4 e 8 dias de exposição aérea retornando aos valores basais ao final do experimento, apesar de não haver diferenças significativas. A glicemia não apresentou diferenças significativas quanto aos estressores aplicados. Os parâmetros hematológicos da série branca, principalmente, o número de linfócitos, neutrófilos e eosinófilos mostraram diferença significativa aos 12 dias de experimentação quando comparados com o momento zero; concluindo-se que, em condições controladas, o adensamento de até 10 girinos/litro e a exposição aérea por 15 minutos não apresentou danos aos girinos de rã-touro durante o período experimental. O padrão de resposta a estes estímulos talvez seja expresso em outro nível hormonal (corticosterona).

Porous polyurethane coatings bonded onto surface-modified titanium substrates for the growth of an endothelial cell layer

Cardiovascular diseases refer to the class of diseases that involve the heart or blood vessels (arteries and veins). Examples of medical devices for treating the cardiovascular diseases include ventricular assist devices (VADs), artificial heart valves and stents. Metallic biomaterials such as titanium and its alloy are commonly used for ventricular assist devices. However, titanium and its alloy show unacceptable thrombosis, which represents a major obstacle to be overcome. Polyurethane (PU) polymer has better blood compatibility and has been used widely in cardiovascular devices. Thus one aim of the project was to coat a PU polymer onto a titanium substrate by increasing the surface roughness, and surface functionality. Since the endothelium of a blood vessel has the most ideal non-thrombogenic properties, it was the target of this research project to grow an endothelial cell layer as a biological coating based on the tissue engineering strategy. However, seeding endothelial cells on the smooth PU coating surfaces is problematic due to the quick loss of seeded cells which do not adhere to the PU surface. Thus it was another aim of the project to create a porous PU top layer on the dense PU pre-layer-coated titanium substrate. The method of preparing the porous PU layer was based on the solvent casting/particulate leaching (SCPL) modified with centrifugation. Without the step of centrifugation, the distribution of the salt particles was not uniform within the polymer solution, and the degree of interconnection between the salt particles was not well controlled. Using the centrifugal treatment, the pore distribution became uniform and the pore interconnectivity was improved even at a high polymer solution concentration (20%) as the maximal salt weight was added in the polymer solution. The titanium surfaces were modified by alkli and heat treatment, followed by functionlisation using hydrogen peroxide. A silane coupling agent was coated before the application of the dense PU pre-layer and the porous PU top layer. The ability of the porous top layer to grow and retain the endothelial cells was also assessed through cell culture techniques. The bonding strengths of the PU coatings to the modified titanium substrates were measured and related to the surface morphologies. The outcome of the project is that it has laid a foundation to achieve the strategy of endothelialisation for the blood compatibility of medical devices. This thesis is divided into seven chapters. Chapter 2 describes the current state of the art in the field of surface modification in cardiovascular devices such as ventricular assist devices (VADs). It also analyses the pros and cons of the existing coatings, particularly in the context of this research. The surface coatings for VADs have evolved from early organic/ inorganic (passive) coatings, to bioactive coatings (e.g. biomolecules), and to cell-based coatings. Based on the commercial applications and the potential of the coatings, the relevant review is focused on the following six types of coatings: (1) titanium nitride (TiN) coatings, (2) diamond-like carbon (DLC) coatings, (3) 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer coatings, (4) heparin coatings, (5) textured surfaces, and (6) endothelial cell lining. Chapter 3 reviews the polymer scaffolds and one relevant fabrication method. In tissue engineering, the function of a polymeric material is to provide a 3-dimensional architecture (scaffold) which is typically used to accommodate transplanted cells and to guide their growth and the regeneration of tissue. The success of these systems is dependent on the design of the tissue engineering scaffolds. Chapter 4 describes chemical surface treatments for titanium and titanium alloys to increase the bond strength to polymer by altering the substrate surface, for example, by increasing surface roughness or changing surface chemistry. The nature of the surface treatment prior to bonding is found to be a major factor controlling the bonding strength. By increasing surface roughness, an increase in surface area occurs, which allows the adhesive to flow in and around the irregularities on the surface to form a mechanical bond. Changing surface chemistry also results in the formation of a chemical bond. Chapter 5 shows that bond strengths between titanium and polyurethane could be significantly improved by surface treating the titanium prior to bonding. Alkaline heat treatment and H2O2 treatment were applied to change the surface roughness and the surface chemistry of titanium. Surface treatment increases the bond strength by altering the substrate surface in a number of ways, including increasing the surface roughness and changing the surface chemistry. Chapter 6 deals with the characterization of the polyurethane scaffolds, which were fabricated using an enhanced solvent casting/particulate (salt) leaching (SCPL) method developed for preparing three-dimensional porous scaffolds for cardiac tissue engineering. The enhanced method involves the combination of a conventional SCPL method and a step of centrifugation, with the centrifugation being employed to improve the pore uniformity and interconnectivity of the scaffolds. It is shown that the enhanced SCPL method and a collagen coating resulted in a spatially uniform distribution of cells throughout the collagen-coated PU scaffolds.In Chapter 7, the enhanced SCPL method is used to form porous features on the polyurethane-coated titanium substrate. The cavities anchored the endothelial cells to remain on the blood contacting surfaces. It is shown that the surface porosities created by the enhanced SCPL may be useful in forming a stable endothelial layer upon the blood contacting surface. Chapter 8 finally summarises the entire work performed on the fabrication and analysis of the polymer-Ti bonding, the enhanced SCPL method and the PU microporous surface on the metallic substrate. It then outlines the possibilities for future work and research in this area.

Expression of chondromodulin-1 in the temporomandibular joint condylar cartilage and disc

BACKGROUND: The temporomandibular joint (TMJ) cartilage consists of condylar cartilage and disc and undergoes continuous remodeling throughout post-natal life. To maintain the integrity of the TMJ cartilage, anti-angiogenic factors play an important role during the remodeling process. In this study, we investigated the expression of the anti-angiogenic factor, chondromodulin- 1 (ChM-1), in TMJ cartilage and evaluate its potential role in TMJ remodeling. METHODS: Eight TMJ specimens were collected from six 4-month-old Japanese white rabbits. Safranin-O staining was performed to determine proteoglycan content. ChM-1 expression in TMJ condylar cartilage and disc was determined by immunohistochemistry. Three human perforated disc tissue samples were collected for investigation of ChM-1 and vascular endothelial growth factor (VEGF) distribution in perforated TMJ disc. RESULTS: Safranin-O stained weakly in TMJ compared with tibial articular and epiphyseal cartilage. In TMJ, ChM-1 was expressed in the proliferative and hypertrophic zone of condylar cartilage and chondrocyte-like cells in the disc. No expression of ChM-1 was observed in osteoblasts and subchondral bone. ChM-1 and VEGF were both similarly expressed in perforated disc tissues. CONCLUSIONS: ChM-1 may play a role in the regulation of TMJ remodeling by preventing blood vessel invasion of the cartilage, thereby maintaining condylar cartilage and disc integrity.