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Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

University Medical Center Hamburg-Eppendorf and DZHK (German Center for Cardiovascular Research)

The CEA-related cell adhesion molecule 1, CEACAM1, is a glycoprotein expressed on the surface of human granulocytes and lymphocytes, endothelia, and many epithelia. CEACAM1 is involved in the regulation of important biological processes, such as tumor growth, angiogenesis, and modulation of the immune response. CEACAM1, a member of the immunoglobulin superfamily carries several Lewis x (Lex) structures as we recently demonstrated by mass spectrometry of native CEACAM1 from human granulocytes. Since Lex residues of pathogens bind to the C-type lectin dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) expressed on human DCs, we hypothesized that Lex glycans of CEACAM1 are recognized by DC-SIGN. Here, we demonstrate that CEACAM1, the major carrier of Lex residues in human granulocytes, is specifically recognized by DC-SIGN via Lex residues mediating the internalization of CEACAM1 into immature DCs. Expression studies with CEACAM1 in combination with different fucosyltransferases (FUTs) revealed that FUTIX plays a key role in the synthesis of Lex groups of CEACAM1. As Lex groups on CEACAM1 are selectively attached and specifically interact with DC-SIGN, our findings suggest that CEACAM1 participates in immune regulation in physiological conditions and in pathological conditions, such as inflammation, autoimmune disease, and cancer.

CEACAM1, an adhesion molecule of human granulocytes, is fucosylated by fucosyltransferase IX and interacts with DC-SIGN of dendritic cells via Lewis x residues.

Intercellular adhesion molecule-3 (ICAM-3) binds to the alpha(L)beta(2) integrin and mediates the contact between T cells and antigen-presenting cells. It has been suggested that dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN), a C-type lectin of macrophages and DCs, is an additional ligand of ICAM-3. So far, the glycan structure mediating the interaction of native ICAM-3 with DC-SIGN is undefined. Here, we demonstrate that native ICAM-3 from human peripheral leukocytes binds recombinant DC-SIGN, is recognized by monoclonal Lewis x antibodies, and specifically interacts with DC-SIGN on immature DCs. The presence of Lewis x residues on ICAM-3 was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy. Investigations on different peripheral blood cell populations revealed that only ICAM-3 from granulocytes bound DC-SIGN. Cotransfection studies demonstrated that fucosyltransferase (FUT) IX and, to a significantly lesser extent, FUT IV, but not FUTs III and VII, mediate the synthesis of Lewis x residues on ICAM-3. These findings indicate that FUT IX is the main FUT mediating the synthesis of Lewis x residues of ICAM-3 in cells of the myeloid lineage, and that these residues bind DC-SIGN. The results suggest that ICAM-3 assists in the interaction of granulocytes with DC-SIGN of DCs.

DC-SIGN binds ICAM-3 isolated from peripheral human leukocytes through Lewis x residues.

Members of the family of carcinoembryonic antigen (CEA)-related cell adhesion molecules (CEACAMs) belonging to the immunoglobulin (Ig) superfamily are expressed in a variety of normal and malignant human tissues. As components of the cell membrane, these glycoproteins can make contact with adjacent cells. CEACAM1 and CEACAM5 (CEA) express Lewis(x) (Le(x)) structures. As shown by mass spectrometry in conjunction with enzymatic digestion, CEACAM1 contains at least seven Le(x) residues. Fucosyltransferase IX is the main fucosyltransferase responsible for attachment of terminal fucose, the key feature of the Le(x) structure, to CEA and CEACAM1. The Le(x) residues of both, CEACAM1 and CEA, interact with the human Le(x)-binding glycan receptors DC-SIGN and SRCL. Since subpopulations of human macrophages express DC-SIGN or SRCL, Le(x)-carrying CEACAMs may modulate the immune response in normal tissues such as the human placenta or in malignant tumours, for example in colorectal, pancreatic or lung carcinomas.

DC-SIGN and SRCL bind glycans of carcinoembryonic antigen (CEA) and CEA-related cell adhesion molecule 1 (CEACAM1): recombinant human glycan-binding receptors as analytical tools.

Specialized protein domains bind to posttranslational modifications (PTMs) of proteins, such as phosphorylation or glycosylation. When such PTM-binding protein domains are used as analytical tools, the functional states of cells and tissues can be determined with high precision. Here, we describe the use of recombinant CLEC10A (CD301), a human glycoreceptor of the C-type lectin family, for the detection of ligands in sections from formalin-fixed, paraffin-embedded normal and cancerous mammary tissues. A construct, in which part of the carbohydrate recognition domain (CRD) was deleted, was used as a negative control. In comparison to normal mammary glands, a pronounced staining of tumor tissues was observed. Because the construct with the truncated CRD did not show any tissue staining, the binding of the wild-type glycoreceptor can be attributed to its carbohydrate recognition domain. To distinguish our novel approach from immunohistochemistry, we propose the designation "protein domain histochemistry" (PDH).

Protein domain histochemistry (PDH): binding of the carbohydrate recognition domain (CRD) of recombinant human glycoreceptor CLEC10A (CD301) to formalin-fixed, paraffin-embedded breast cancer tissues.

Analyzing contractile force, the most important and best understood function of cardiomyocytes in vivo is not established in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). This study describes the generation of 3D, strip-format, force-generating engineered heart tissues (EHT) from hiPSC-CM and their physiological and pharmacological properties. CM were differentiated from hiPSC by a growth factor-based three-stage protocol. EHTs were generated and analyzed histologically and functionally. HiPSC-CM in EHTs showed well-developed sarcomeric organization and alignment, and frequent mitochondria. Systematic contractility analysis (26 concentration-response curves) reveals that EHTs replicated canonical response to physiological and pharmacological regulators of inotropy, membrane- and calcium-clock mediators of pacemaking, modulators of ion-channel currents, and proarrhythmic compounds with unprecedented precision. The analysis demonstrates a high degree of similarity between hiPSC-CM in EHT format and native human heart tissue, indicating that human EHTs are useful for preclinical drug testing and disease modeling.

Birgit Klampe

Department of Experimental Pharmacology and Toxicology,
Cardiovascular Research Center,
Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center

University Medical Center Hamburg-Eppendorf and DZHK (German Center for Cardiovascular Research)

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Birgit Klampe

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Experimental Pharmacology and Toxicology,Cardiovascular Research Center,Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center

University Medical Center Hamburg-Eppendorf and DZHK (German Center for Cardiovascular Research)

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Department of Experimental Pharmacology and Toxicology,
Cardiovascular Research Center,
Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center