Christian Frie

The Targeted Deletion of the LAMC1 Gene

Laminin is required for the production of a basement membrane in both the early embryo and in the embryoid body. The absence of the gamma 1 chain leads to different processing of the other subunits in the trimer and prevents the production of a polymerizing laminin molecule. Collagen IV and perlecan are not able to compensate for the loss of the laminin molecule, and nidogen, which would normally bind to the gamma 1 chain, is almost completely lost from the embryoid body. In the embryo the failure of the basement membrane results in embryonic lethality at embryonic day 5.5, a time when the primitive endoderm is differentiating to visceral and parietal endoderm, and the latter is migrating out of the inner cell mass over the trophectoderm. It is likely that failure in one or both of these events leads to the death of the embryo. The absence of the trophectodermal basement membrane normally present in the blastocyst appears not to be necessary for the formation of the blastocystic cavity, which depends on the formation of a polarized epithelium. Hence the first epithelium produced in the developing embryo does so independently of a basement membrane.

Expression of matrilin-1, -2 and -3 in developing mouse limbs and heart.

The expression of matrilin-1, -2 and -3 was studied in the heart and limb during mouse development. Matrilin-1 is transiently expressed in the heart between days 9.5 and 14.5 p.c. Matrilin-2 expression was detected in the heart from day 10.5 p.c. onwards. In the developing limb bud, both matrilin-1 and -3 were observed first at day 12.5 p.c. Throughout development matrilin-3 expression was strictly limited to cartilage, while matrilin-1 was also found in some other forms of connective tissue. Matrilin-2, albeit present around hypertrophic chondrocytes in the growth plate, was mainly expressed in non-skeletal structures. The complementary, but in part overlapping, expression of matrilins indicates the possibility for both redundant and unique functions among the members of this novel family of extracellular matrix proteins.

Identification of Novel Binding Partners (Annexins) for the Cell Death Signal Phosphatidylserine and Definition of Their Recognition Motif

Identification and clearance of apoptotic cells prevents the release of harmful cell contents thereby suppressing inflammation and autoimmune reactions. Highly conserved annexins may modulate the phagocytic cell removal by acting as bridging molecules to phosphatidylserine, a characteristic phagocytosis signal of dying cells. In this study five members of the structurally and functionally related annexin family were characterized for their capacity to interact with phosphatidylserine and dying cells. The results showed that AnxA3, AnxA4, AnxA13, and the already described interaction partner AnxA5 can bind to phosphatidylserine and apoptotic cells, whereas AnxA8 lacks this ability. Sequence alignment experiments located the essential amino residues for the recognition of surface exposed phosphatidylserine within the calcium binding motifs common to all annexins. These amino acid residues were missing in the evolutionary young AnxA8 and when they were reintroduced by site directed mutagenesis AnxA8 gains the capability to interact with phosphatidylserine containing liposomes and apoptotic cells. By defining the evolutionary conserved amino acid residues mediating phosphatidylserine binding of annexins we show that the recognition of dying cells represent a common feature of most annexins. Hence, the individual annexin repertoire bound to the cell surface of dying cells may fulfil opsonin-like function in cell death recognition.

Epidermal Transglutaminase (TGase 3) Is Required for Proper Hair Development, but Not the Formation of the Epidermal Barrier

Transglutaminases (TGase), a family of cross-linking enzymes present in most cell types, are important in events as diverse as cell-signaling and matrix stabilization. Transglutaminase 1 is crucial in developing the epidermal barrier, however the skin also contains other family members, in particular TGase 3. This isoform is highly expressed in the cornified layer, where it is believed to stabilize the epidermis and its reduction is implicated in psoriasis. To understand the importance of TGase 3 in vivo we have generated and analyzed mice lacking this protein. Surprisingly, these animals display no obvious defect in skin development, no overt changes in barrier function or ability to heal wounds. In contrast, hair lacking TGase 3 is thinner, has major alterations in the cuticle cells and hair protein cross-linking is markedly decreased. Apparently, while TGase 3 is of unique functional importance in hair, in the epidermis loss of TGase 3 can be compensated for by other family members.

Deficiency of annexins A5 and A6 induces complex changes in the transcriptome of growth plate cartilage but does not inhibit the induction of mineralization

Initiation of mineralization during endochondral ossification is a multistep process and has been assumed to correlate with specific interactions of annexins A5 and A6 and collagens. However, skeletal development appears to be normal in mice deficient for either A5 or A6, and the highly conserved structures led to the assumption that A5 and A6 may fulfill redundant functions. We have now generated mice deficient of both proteins. These mice were viable and fertile and showed no obvious abnormalities. Assessment of skeletal elements using histologic, ultrastructural, and peripheral quantitative computed tomographic methods revealed that mineralization and development of the skeleton were not significantly affected in mutant mice. Otherwise, global gene expression analysis showed subtle changes at the transcriptome level of genes involved in cell growth and intermediate metabolism. These results indicate that annexins A5 and A6 may not represent the essential annexins that promote mineralization in vivo.

Depletion of annexin A5, annexin A6, and collagen X causes no gross changes in matrix vesicle–mediated mineralization, but lack of collagen X affects hematopoiesis and the Th1/Th2 response

Numerous biochemical studies have pointed to an essential role of annexin A5 (AnxA5), annexin A6 (AnxA6), and collagen X in matrix vesicle–mediated biomineralization during endochondral ossification and in osteoarthritis. By binding to the extracellular matrix protein collagen X and matrix vesicles, annexins were proposed to anchor matrix vesicles in the extracellular space of hypertrophic chondrocytes to initiate the calcification of cartilage. However, mineralization appears to be normal in mice lacking AnxA5 and AnxA6, whereas collagen X–deficient mice show only subtle alterations in the growth plate organization. We hypothesized that the simultaneous lack of AnxA5, AnxA6, and collagen X in vivo induces more pronounced changes in the growth plate development and the initiation of mineralization. In this study, we generated and analyzed mice deficient for AnxA5, AnxA6, and collagen X. Surprisingly, mice were viable, fertile, and showed no obvious abnormalities. Assessment of growth plate development indicated that the hypertrophic zone was expanded in Col10a1−/− and AnxA5−/−AnxA6−/−Col10a1−/− newborns, whereas endochondral ossification and mineralization were not affected in 13‐day‐ and 1‐month‐old mutants. In peripheral quantitative computed tomography, no changes in the degree of biomineralization were found in femora of 1‐month‐ and 1‐year‐old mutants even though the diaphyseal circumference was reduced in Col10a1−/− and AnxA5−/−AnxA6−/−Col10a1−/− mice. The percentage of naive immature IgM+/IgM+ B cells and peripheral T‐helper cells were increased in Col10a1−/− and AnxA5−/−AnxA6−/−Col10a1−/− mutants, and activated splenic T cells isolated from Col10a1−/− mice secreted elevated levels of IL‐4 and GM‐CSF. Hence, collagen X is needed for hematopoiesis during endochondral ossification and for the immune response, but the interaction of annexin A5, annexin A6, and collagen X is not essential for physiological calcification of growth plate cartilage. Therefore, annexins and collagen X may rather fulfill functions in growth plate cartilage not directly linked to the mineralization process.

Sorting of growth plate chondrocytes allows the isolation and characterization of cells of a defined differentiation status.

Axial growth of long bones occurs through a coordinated process of growth plate chondrocyte proliferation and differentiation. This maturation of chondrocytes is reflected in a zonal change in gene expression and cell morphology from resting to proliferative, prehypertrophic, and hypertrophic chondrocytes of the growth plate followed by ossification. A major experimental limitation in understanding growth plate biology and pathophysiology is the lack of a robust technique to isolate cells from the different zones, particularly from small animals. Here, we report on a new strategy for separating distinct chondrocyte populations from mouse growth plates. By transcriptome profiling of microdissected zones of growth plates, we identified novel, zone‐specific cell surface markers and used these for flow cytometry and immunomagnetic cell separation to quantify, enrich, and characterize chondrocytes populations with respect to their differentiation status. This approach provides a novel platform to study cartilage development and characterize mouse growth plate chondrocytes to reveal unique cellular phenotypes of the distinct subpopulations within the growth plate.

PECAM1+/Sca1+/CD38+ Vascular Cells Transform into Myofibroblast-Like Cells in Skin Wound Repair

Skin injury induces the formation of new blood vessels by activating the vasculature in order to restore tissue homeostasis. Vascular cells may also differentiate into matrix-secreting contractile myofibroblasts to promote wound closure. Here, we characterize a PECAM1+/Sca1+ vascular cell population in mouse skin, which is highly enriched in wounds at the peak of neoangiogenesis and myofibroblast formation. These cells express endothelial and perivascular markers and present the receptor CD38 on their surface. PECAM1+/Sca1+/CD38+ cells proliferate upon wounding and could give rise to α-SMA+ myofibroblast-like cells. CD38 stimulation in immunodeficient mice reduced the wound size at the peak of neoangiogenesis and myofibroblast formation. In humans a corresponding cell population was identified, which was enriched in sprouting vessels of basal cell carcinoma biopsies. The results indicate that PECAM1+/Sca1+/CD38+ vascular cells could proliferate and differentiate into myofibroblast-like cells in wound repair. Moreover, CD38 signaling modulates PECAM1+/Sca1+/CD38+ cell activation in the healing process implying CD38 as a target for anti-angiogenic therapies in human basal cell carcinoma.

miR-126-3p promotes matrix-dependent perivascular cell attachment, migration and intercellular interaction.

microRNAs (miRNAs) can regulate the interplay between perivascular cells (PVC) and endothelial cells (EC) during angiogenesis, but the relevant PVC‐specific miRNAs are not yet defined. Here, we identified miR‐126‐3p and miR‐146a to be exclusively upregulated in PVC upon interaction with EC, determined their influence on the PVC phenotype and elucidate their molecular mechanisms of action. Specifically the increase of miR‐126‐3p strongly promoted the motility of PVC on the basement membrane‐like composite and stabilized networks of EC. Subsequent miRNA target analysis showed that miR‐126‐3p inhibits SPRED1 and PLK2 expression, induces ERK1/2 phosphorylation and stimulates TLR3 expression to modulate cell‐cell and cell‐matrix contacts of PVC. Gain of expression experiments in vivo demonstrated that miR‐126‐3p stimulates PVC coverage of newly formed vessels and transform immature into mature, less permeable vessels. In conclusion we showed that miR‐126‐3p regulates matrix‐dependent PVC migration and intercellular interaction to modulate vascular integrity. Stem Cells 2016;34:1297–1309

Identification of a myofibroblast-specific expression signature in skin wounds

After skin injury fibroblasts migrate into the wound and transform into contractile, extracellular matrix-producing myofibroblasts to promote skin repair. Persistent activation of myofibroblasts can cause excessive fibrotic reactions, but the underlying mechanisms are not fully understood. We used SMA-GFP transgenic mice to study myofibroblast recruitment and activation in skin wounds. Myofibroblasts were initially recruited to wounds three days post injury, their number reached a maximum after seven days and subsequently declined. Expression profiling showed that 1749 genes were differentially expressed in sorted myofibroblasts from wounds seven days post injury. Most of these genes were linked with the extracellular region and cell periphery including genes encoding for extracellular matrix proteins. A unique panel of core matrisome and matrisome-associated genes was differentially expressed in myofibroblasts and several genes not yet known to be linked to myofibroblast-mediated wound healing were found (e.g. Col24a1, Podnl1, Bvcan, Tinagl1, Thbs3, Adamts16, Adamts19, Cxcls, Ccls). In addition, a complex network of G protein-coupled signaling events was regulated in myofibroblasts (e.g. Adcy1, Plbc4, Gnas). Hence, this first characterization of a myofibroblast-specific expression profile at the peak of in situ granulation tissue formation provides important insights into novel target genes that may control excessive ECM deposition during fibrotic reactions.