Effect of N-Acetylcysteine on Adipose-Derived Stem Cell and Autologous Fat Graft Survival in a Mouse Model.

BACKGROUND: Autologous fat grafting is a popular reconstructive technique, but is limited by inconsistent graft retention. The authors examined whether a widely available, clinically safe antioxidant, N-acetylcysteine, could improve adipose-derived stem cell survival and graft take when added to tumescent solution during fat harvest. METHODS: Inguinal fat pads were harvested from C57BL/6 mice using tumescent solution with or without N-acetylcysteine. Flow cytometric, proliferation, and differentiation assays were performed on isolated primary adipose-derived stem cells and 3T3-L1 preadipocytes treated with or without hydrogen peroxide and/or N-acetylcysteine. N-Acetylcysteine-treated or control grafts were injected under recipient mouse scalps and assessed by serial micro-computed tomographic volumetric analysis. Explanted grafts underwent immunohistochemical analysis. RESULTS: In culture, N-acetylcysteine protected adipose-derived stem cells from oxidative stress and improved cell survival following hydrogen peroxide treatment. Combined exposure to both N-acetylcysteine and hydrogen peroxide led to a 200-fold increase in adipose-derived stem cell proliferation, significantly higher than with either agent alone. N-Acetylcysteine decreased differentiation of adipose-derived stem cells into mature adipocytes, as evidenced by decreased transcription of adipocyte differentiation markers and reduced Oil Red-O staining. In vivo, N-acetylcysteine treatment resulted in improved graft retention at 3 months compared with control (46 versus 17 percent; p = 0.027). N-Acetylcysteine-treated grafts demonstrated less fibrosis and inflammation, and a 33 percent increase in adipocyte density compared with controls (p < 0.001) that was not associated with increased vascularity. CONCLUSION: These findings provide proof of principle for the addition of N-acetylcysteine to tumescent harvest solution in the clinical setting to optimize fat graft yields.

Alternatively activated M2 macrophages improve autologous Fat Graft survival in a mouse model through induction of angiogenesis.

BACKGROUND: Variability in graft retention with subsequent undercorrection remains a significant limitation of autologous fat grafting. The authors evaluated whether graft retention in a mouse model could be improved via graft supplementation with alternatively activated M2 macrophages, cells known to play a critical role in tissue repair. METHODS: Grafts from C57BL/6 mouse inguinal fat pads were supplemented with M2 macrophages generated by intraperitoneal Brewer's thioglycollate injection and in vitro culture. Grafts with saline or M2 macrophages were injected under recipient mouse scalps and assessed by serial micro-computed tomographic analysis. Explanted grafts underwent immunohistochemical and flow cytometric analyses. M2 culture supernatants were added to stromal vascular fraction adipose-derived stem cells to assess adipogenic gene expression induction. RESULTS: One month after graft injection, no significant difference was noted between M2 macrophage-supplemented (105 ± 7.0 mm) and control graft volumes (72 ± 22 mm). By 3 months after injection, M2 macrophage-supplemented grafts remained stable, whereas controls experienced further volume loss (103 ± 8 mm versus 39.4 ± 15 mm; p = 0.015). Presence of macrophages in supplemented grafts was confirmed by flow cytometry. M2 macrophage-supplemented grafts exhibited a 157 percent increase in vascular density compared with controls (p < 0.05). Induction of adipogenic C/EBPα gene expression was observed with M2 supernatants addition to stromal vascular fraction adipose-derived stem cells. CONCLUSIONS: M2 macrophages improve autologous fat graft volume retention by stimulating angiogenesis. These findings provide proof-of-principle for development of fat grafting techniques that harness reparative properties of M2 macrophages.

Plasminogen receptor S100A10 is essential for the migration of tumor-promoting macrophages into tumor sites.

Macrophages are critical drivers of tumor growth, invasion, and metastasis. Movement of macrophages into tumors requires the activity of cell surface proteases such as plasmin. In this study, we offer genetic evidence that plasminogen receptor S100A10 is essential for recruitment of macrophages to the tumor site. Growth of murine Lewis lung carcinomas or T241 fibrosarcomas was dramatically reduced in S100A10-deficient mice compared with wild-type mice. The tumor growth deficit corresponded with a decrease in macrophage density that could be rescued by intraperitoneal injection of wild-type but not S100A10-deficient macrophages. Notably, macrophages of either genotype could rescue tumor growth if they were injected into the tumor itself, establishing that S100A10 was required specifically for the migratory capability needed for tumor homing. Conversely, selective depletion of macrophages from wild-type mice phenocopied the tumor growth deficit seen in S100A10-deficient mice. Together, our findings show that S100A10 is essential and sufficient for macrophage migration to tumor sites, and they define a novel rate-limiting step in tumor progression.

The role of the annexin A2 heterotetramer in vascular fibrinolysis.

The vascular endothelial cells line the inner surface of blood vessels and function to maintain blood fluidity by producing the protease plasmin that removes blood clots from the vasculature, a process called fibrinolysis. Plasminogen receptors play a central role in the regulation of plasmin activity. The protein complex annexin A2 heterotetramer (AIIt) is an important plasminogen receptor at the surface of the endothelial cell. AIIt is composed of 2 molecules of annexin A2 (ANXA2) bound together by a dimer of the protein S100A10. Recent work performed by our laboratory allowed us to clarify the specific roles played by ANXA2 and S100A10 subunits within the AIIt complex, which has been the subject of debate for many years. The ANXA2 subunit of AIIt functions to stabilize and anchor S100A10 to the plasma membrane, whereas the S100A10 subunit initiates the fibrinolytic cascade by colocalizing with the urokinase type plasminogen activator and receptor complex and also providing a common binding site for both tissue-type plasminogen activator and plasminogen via its C-terminal lysine residue. The AIIt mediated colocalization of the plasminogen activators with plasminogen results in the rapid and localized generation of plasmin to the endothelial cell surface, thereby regulating fibrinolysis.

Regulation of fibrinolysis by S100A10 in vivo.

Endothelial cells form the inner lining of vascular networks and maintain blood fluidity by inhibiting blood coagulation and promoting blood clot dissolution (fibrinolysis). Plasmin, the primary fibrinolytic enzyme, is generated by the cleavage of the plasma protein, plasminogen, by its activator, tissue plasminogen activator. This reaction is regulated by plasminogen receptors at the surface of the vascular endothelial cells. Previous studies have identified the plasminogen receptor protein S100A10 as a key regulator of plasmin generation by cancer cells and macrophages. Here we examine the role of S100A10 and its annexin A2 binding partner in endothelial cell function using a homozygous S100A10-null mouse. Compared with wild-type mice, S100A10-null mice displayed increased deposition of fibrin in the vasculature and reduced clearance of batroxobin-induced vascular thrombi, suggesting a role for S100A10 in fibrinolysis in vivo. Compared with wild-type cells, endothelial cells from S100A10-null mice demonstrated a 40% reduction in plasminogen binding and plasmin generation in vitro. Furthermore, S100A10-deficient endothelial cells demonstrated impaired neovascularization of Matrigel plugs in vivo, suggesting a role for S100A10 in angiogenesis. These results establish an important role for S100A10 in the regulation of fibrinolysis and angiogenesis in vivo, suggesting S100A10 plays a critical role in endothelial cell function.

NUCLEOMORPH KARYOTYPE DIVERSITY IN THE FRESHWATER CRYPTOPHYTE GENUS CRYPTOMONAS(1).

Cryptophytes are unicellular, biflagellate algae with plastids (chloroplasts) derived from the uptake of a red algal endosymbiont. These organisms are unusual in that the nucleus of the engulfed red alga persists in a highly reduced form called a nucleomorph. Nucleomorph genomes are remarkable in their small size (<1,000 kilobase pairs [kbp]) and high degree of compaction (∼1 kbp per gene). Here, we investigated the molecular and karyotypic diversity of nucleomorph genomes in members of the genus Cryptomonas. 18S rDNA genes were amplified, sequenced, and analyzed from C. tetrapyrenoidosa Skuja CCAP979/63, C. erosa Ehrenb. emmend. Hoef-Emden CCAP979/67, Cryptomonas sp. CCAP979/52, C. lundii Hoef-Emden et Melkonian CCAP979/69, and C. lucens Skuja CCAP979/35 in the context of a large set of publicly available nucleomorph 18S rDNA sequences. Pulsed-field gel electrophoresis (PFGE) was used to examine the nucleomorph genome karyotype of each of these strains. Individual chromosomes ranged from ∼160 to 280 kbp in size, with total genome sizes estimated to be ∼600-655 kbp. Unexpectedly, the nucleomorph karyotype of Cryptomonas sp. CCAP979/52 is significantly different from that of C. tetrapyrenoidosa and C. lucens, despite the fact that their 18S rDNA genes are >99% identical to one another. These results suggest that nucleomorph karyotype similarity is not a reliable indicator of evolutionary affinity and provides a starting point for further investigation of the fine-scale dynamics of nucleomorph genome evolution within members of the genus Cryptomonas.