Direct reprogramming of fibroblasts into endothelial cells capable of angiogenesis and reendothelialization in tissue-engineered vessels

The generation of induced pluripotent stem (iPS) cells is an important tool for regenerative medicine. However, the main restriction is the risk of tumor development. In this study we found that during the early stages of somatic cell reprogramming toward a pluripotent state, specific gene expression patterns are altered. Therefore, we developed a method to generate partial-iPS (PiPS) cells by transferring four reprogramming factors (OCT4, SOX2, KLF4, and c-MYC) to human fibroblasts for 4 d. PiPS cells did not form tumors in vivo and clearly displayed the potential to differentiate into endothelial cells (ECs) in response to defined media and culture conditions. To clarify the mechanism of PiPS cell differentiation into ECs, SET translocation (myeloid leukemia-associated) (SET) similar protein (SETSIP) was indentified to be induced during somatic cell reprogramming. Importantly, when PiPS cells were treated with VEGF, SETSIP was translocated to the cell nucleus, directly bound to the VE-cadherin promoter, increasing vascular endothelial-cadherin (VE-cadherin) expression levels and EC differentiation. Functionally, PiPS-ECs improved neovascularization and blood flow recovery in a hindlimb ischemic model. Furthermore, PiPS-ECs displayed good attachment, stabilization, patency, and typical vascular structure when seeded on decellularized vessel scaffolds. These findings indicate that reprogramming of fibroblasts into ECs via SETSIP and VEGF has a potential clinical application.

Smooth Muscle Cells Differentiated from Reprogrammed Embryonic Lung Fibroblasts Through DKK3 Signalling are Potent for Tissue Engineering of Vascular Grafts

Rationale: Smooth muscle cells (SMCs) are a key component of tissue-engineered vessels. However, the sources by which they can be isolated are limited.

Objective: We hypothesized that a large number of SMCs could be obtained by direct reprogramming of fibroblasts, that is, direct differentiation of specific cell lineages before the cells reaching the pluripotent state.

Methods and Results: We designed a combined protocol of reprogramming and differentiation of human neonatal lung fibroblasts. Four reprogramming factors (OCT4, SOX2, KLF4, and cMYC) were overexpressed in fibroblasts under reprogramming conditions for 4 days with cells defined as partially-induced pluripotent stem (PiPS) cells. PiPS cells did not form tumors in vivo after subcutaneous transplantation in severe combined immunodeficiency mice and differentiated into SMCs when seeded on collagen IV and maintained in differentiation media. PiPS-SMCs expressed a panel of SMC markers at mRNA and protein levels. Furthermore, the gene dickkopf 3 was found to be involved in the mechanism of PiPS-SMC differentiation. It was revealed that dickkopf 3 transcriptionally regulated SM22 by potentiation of Wnt signaling and interaction with Kremen1. Finally, PiPS-SMCs repopulated decellularized vessel grafts and ultimately gave rise to functional tissue-engineered vessels when combined with previously established PiPS-endothelial cells, leading to increased survival of severe combined immunodeficiency mice after transplantation of the vessel as a vascular graft.

Conclusions: We developed a protocol to generate SMCs from PiPS cells through a dickkopf 3 signaling pathway, useful for generating tissue-engineered vessels. These findings provide a new insight into the mechanisms of SMC differentiation with vast therapeutic potential.

Developing mRNA-based biomarkers from formalin-fixed paraffin-embedded tissue

Cancer is a complex and heterogeneous disease which is one of the leading causes of death in Western civilisations. Thus, oncology is viewed as a primary focus for personalized medicine. It is recognised that cancer treatment needs to be better tailored in order to improve patient outcome. Patient tumor samples will be required to characterize cancer at a molecular level and identify where there may be disease subgroups that should be treated differently. The use of formalin-fixed paraffin-embedded tissue is important for enabling such studies. In this report, we focus on the challenges that have been faced to date along with the technological developments that have now made this possible. We also highlight the impact this may have on drug and diagnostic development.

Lymphocyte subsets in conjunctival mucosa-associated-lymphoid-tissue after exposure to retinal-S-antigen

Purpose: To evaluate the immune cell subsets in conjunctival mucosa-associated-lymphoid-tissue (C-MALT) following challenge with antigen. Methods: Ten adult female Lewis rats were studied. Five rats received one drop (5 µL) of retinal S-antigen (500 µg/mL in phosphate buffered saline, PBS) instilled into the lower fornix twice daily for 10 consecutive days. Five rats received PBS only and served as controls for the experiment. Two days after the last instillation the animals were sacrificed and the orbital contents prepared for immunohistological staining. A panel of monoclonal antibodies was used: CD5, CD4, CD8, CD25, and CD45RA. The number of positive cells were counted in sections of epibulbar, forniceal, and tarsal conjunctiva. Results: There was a significant increase in the number of CD8 T lymphocytes in the conjunctiva of animals receiving retinal S-antigen when compared to control animals. Conclusion: Conjunctival instillation of retinal S-antigen causes an immune response in the C-MALT with a significant increase in the CD8 T lymphocyte subset in this tissue. This response may be involved in the induction of tolerance to the encountered antigen.

Fasciola hepatica: A light and electron microscope study of sustentacular tissue and heterophagy in the testis

In order to investigate cytolytic activity in the testis of Fasciola hepatica, flukes belonging to several different triclabendazole (TCBZ)-sensitive and TCBZ-resistant isolates, and wildtype flukes from field infections, were studied by light and electron microscopy with a view to identifying sites of heterophagy and macromolecular hydrolysis. At the periphery of the testis tubules in all the flukes examined, large euchromatic nuclei, each bearing a prominent nucleolus, were seen. These were invested with a thin cytoplasmic layer, extensions of which partially enveloped and probably supported the neighbouring spermatogonia. No lateral cell boundaries were identified in this tissue, possibly indicating syncytial organisation. The tissue, considered to be analogous to Sertoli cells in vertebrate testis, was identified as sustentacular tissue. It displayed cytoplasmic features consistent with protein/glycoprotein synthesis (through a granular endoplasmic reticulum-Golgi mediated mechanism) and intracellular digestion/heterophagy (through a lysosomal system). The intracytoplasmic hydrolytic activity of the sustentacular tissue probably serves to scavenge effete cells and cytoplasmic debris, to recycle useful molecules, to promote spermatozoon maturation and possibly to aid osmoregulation within the tubules. Certain protein-containing macromolecules synthesised in the sustentacular tissue may contribute to the seminiferous fluid, or have pheromonal activity. The presence of numerous mitochondria and abundant smooth endoplasmic reticulum is consistent with facilitation of inward and outward movement of micromolecular nutrients, metabolites including excretory products and water. In the sustentacular tissue of certain flukes with dysfunctional spermiogenesis, there was increased heterophagic and cytolytic scavenging activity. The cytoplasmic residual vacuoles remaining after the release of spermatids were also identified as possible sites for lysosome-mediated intracellular digestion and osmoregulation in the testis tubules of F. hepatica. (C) 2012 Elsevier B.V. All rights reserved.

Guidelines and considerations for conducting experiments using tissue microarrays

Tissue microarrays (TMAs) represent a powerful method for undertaking large-scale tissue-based biomarker studies. While TMAs offer several advantages, there are a number of issues specific to their use which need to be considered when employing this method. Given the investment in TMA-based research, guidance on design and execution of experiments will be of benefit and should help researchers new to TMA-based studies to avoid known pitfalls. Furthermore, a consensus on quality standards for TMA-based experiments should improve the robustness and reproducibility of studies, thereby increasing the likelihood of identifying clinically useful biomarkers. In order to address these issues, the National Cancer Research Institute Biomarker and Imaging Clinical Studies Group organized a 1-day TMA workshop held in Nottingham in May 2012. The document herein summarizes the conclusions from the workshop. It includes guidance and considerations on all aspects of TMA-based research, including the pre-analytical stages of experimental design, the analytical stages of data acquisition, and the postanalytical stages of data analysis. A checklist is presented which can be used both for planning a TMA experiment and interpreting the results of such an experiment. For studies of cancer biomarkers, this checklist could be used as a supplement to the REMARK guidelines.

Development of a Portable Tissue Micro Array Instrument

Tissue micro array (TMA) is based on the idea of applying miniaturization and a high throughput approach to hybridization-based analyses of tissues. It facilitates biomedical research on a large scale in a single experiment; thus representing one of the most commonly used technologies in translational research. A critical analysis of the existing TMA instruments indicates that there are potential constraints in terms of portability, apart from costs and complexity. This paper will present the development of an affordable, configurable, and portable TMA instrument to allow an efficient collection of tissues, especially in instrument-to-tissue scenarios. The purely mechanical instrument requires no energy sources other than the user, is light weight, portable, and simple to use. [DOI: 10.1115/1.4004922]

Biomarkers in diabetes:hemoglobin A1c, vascular and tissue markers

Biomarkers are conventionally defined as "biological molecules that represent health and disease states." They typically are measured in readily available body fluids (blood or urine), lie outside the causal pathway, are able to detect subclinical disease, and are used to monitor clinical and subclinical disease burden and response to treatments. Biomarkers can be "direct" endpoints of the disease itself, or "indirect" or surrogate endpoints. New technologies (such as metabolomics, proteomics, genomics) bring a wealth of opportunity to develop new biomarkers. Other new technologies enable the development of nonmolecular, functional, or biophysical tissue-based biomarkers. Diabetes mellitus is a complex disease affecting almost every tissue and organ system, with metabolic ramifications extending far beyond impaired glucose metabolism. Biomarkers may reflect the presence and severity of hyperglycemia (ie, diabetes itself) or the presence and severity of the vascular complications of diabetes. Illustrative examples are considered in this brief review. In blood, hemoglobin A1c (HbA1c) may be considered as a biomarker for the presence and severity of hyperglycemia, implying diabetes or prediabetes, or, over time, as a "biomarker for a risk factor," ie, hyperglycemia as a risk factor for diabetic retinopathy, nephropathy, and other vascular complications of diabetes. In tissues, glycation and oxidative stress resulting from hyperglycemia and dyslipidemia lead to widespread modification of biomolecules by advanced glycation end products (AGEs). Some of these altered species may serve as biomarkers, whereas others may lie in the causal pathway for vascular damage. New noninvasive technologies can detect tissue damage mediated by AGE formation: these include indirect measures such as pulse wave analysis (a marker of vascular dysfunction) and more direct markers such as skin autofluorescence (a marker of long-term accumulation of AGEs). In the future, we can be optimistic that new blood and tissue-based biomarkers will enable the detection, prevention, and treatment of diabetes and its complications long before overt disease develops.

Oxidised, glycated LDL selectively influences tissue inhibitor of metalloproteinase-3 gene expression and protein production in human retinal capillary pericytes

Aims/hypothesis: Matrix metalloproteinases (MMPs) and their natural inhibitors, tissue inhibitor of metalloproteinases (TIMPs), regulate important biological processes including the homeostasis of the extracellular matrix, proteolysis of cell surface proteins, proteinase zymogen activation, angiogenesis and inflammation. Studies have shown that their balance is altered in retinal microvascular tissues in diabetes. Since LDLs modified by oxidation/glycation are implicated in the pathogenesis of diabetic vascular complications, we examined the effects of modified LDL on the gene expression and protein production of MMPs and TIMPs in retinal pericytes. Methods: Quiescent human retinal pericytes were exposed to native LDL (N-LDL), glycated LDL (G-LDL) and heavily oxidised and glycated LDL (HOG-LDL) for 24 h. We studied the expression of the genes encoding MMPs and TIMPs mRNAs by analysis of microarray data and quantitative PCR, and protein levels by immunoblotting and ELISA. Results: Microarray analysis showed that MMP1, MMP2, MMP11, MMP14 and MMP25 and TIMP1, TIMP2, TIMP3 and TIMP4 were expressed in pericytes. Of these, only TIMP3 mRNA showed altered regulation, being expressed at significantly lower levels in response to HOG- vs N-LDL. Quantitative PCR and immunoblotting of cell/matrix proteins confirmed the reduction in TIMP3 mRNA and protein in response to HOG-LDL. In contrast to cellular TIMP3 protein, analysis of secreted TIMP1, TIMP2, MMP1 and collagenase activity indicated no changes in their production in response to modified LDL. Combined treatment with N- and HOG-LDL restored TIMP3 mRNA expression to a level comparable with that after N-LDL alone. Conclusions/interpretation: Among the genes encoding for MMPs and TIMPs expressed in retinal pericytes, TIMP3 is uniquely regulated by HOG-LDL. Reduced TIMP3 expression might contribute to microvascular abnormalities in diabetic retinopathy. © 2007 Springer-Verlag.