Functions In Tissue Remodeling Research Articles

Exploring the Potential of Micro-Immunotherapy in the Treatment of Periodontitis.

Periodontitis, characterized by the progressive destruction of dental support tissues due to altered immune responses, poses a significant concern for public health. This condition involves intricate interactions between the immune response and oral microbiome, where innate and adaptive immune responses, with their diverse cell populations and inflammatory mediators, play crucial roles in this immunopathology. Indeed, cytokines, chemokines, growth factors, and immune cells perform key functions in tissue remodeling. Focusing on periodontal therapies, our attention turns to micro-immunotherapy (MI), employing low doses (LDs) and ultra-low doses (ULDs) of immunological signaling molecules like cytokines, growth factors, and hormones. Existing studies across various fields lay the groundwork for the application of MI in periodontitis, highlighting its anti-inflammatory and regenerative potential in soft tissue models based on in vitro research. In summary, this review underscores the versatility and potential of MI in managing periodontal health, urging further investigations to solidify its clinical integration. MI supports an innovative approach by modulating immune responses at low doses to address periodontitis.

Roles for epithelial integrin α3β1 in regulation of the microenvironment during normal and pathological tissue remodeling.

Integrin receptors for the extracellular matrix activate intracellular signaling pathways that are critical for tissue development, homeostasis, and regeneration/repair, and their loss or dysregulation contributes to many developmental defects and tissue pathologies. This review will focus on tissue remodeling roles for integrin α3β1, a receptor for laminins found in the basement membranes (BMs) that underlie epithelial cell layers. As a paradigm, we will discuss literature that supports a role for α3β1 in promoting ability of epidermal keratinocytes to modify their tissue microenvironment during skin development, wound healing, or tumorigenesis. Preclinical and clinical studies have shown that this role depends largely on ability of α3β1 to govern the keratinocyte's repertoire of secreted proteins, or the "secretome," including 1) matrix proteins and proteases involved in matrix remodeling and 2) paracrine-acting growth factors/cytokines that stimulate other cells with important tissue remodeling functions (e.g., endothelial cells, fibroblasts, inflammatory cells). Moreover, α3β1 signaling controls gene expression that helps epithelial cells carry out these functions, including genes that encode secreted matrix proteins, proteases, growth factors, or cytokines. We will review what is known about α3β1-dependent gene regulation through both transcription and posttranscriptional mRNA stability. Regarding the latter, we will discuss examples of α3β1-dependent alternative splicing (AS) or alternative polyadenylation (APA) that prevents inclusion of cis-acting mRNA sequences that would otherwise target the transcript for degradation via nonsense-mediated decay or destabilizing AU-rich elements (AREs) in the 3'-untranslated region (3'-UTR). Finally, we will discuss prospects and anticipated challenges of exploiting α3β1 as a clinical target for the treatment of cancer or wound healing.

Papain-Based Solubilization of Decellularized Extracellular Matrix for the Preparation of Bioactive, Thermosensitive Pregels.

Solubilized, gel-forming decellularized extracellular matrix (dECM) is used in a wide range of basic and translational research and due to its inherent bioactivity can promote structural and functional tissue remodeling. The animal-derived protease pepsin has become the standard proteolytic enzyme for the solubilization of almost all types of collagen-based dECM. In this study, pepsin was compared with papain, α-amylase, and collagenase for their potential to solubilize porcine liver dECM. Maximum preservation of bioactive components and native dECM properties was used as a decisive criterion for further application of the enzymes, with emphasis on minimal destruction of the protein structure and maintained capacity for physical thermogelation at neutral pH. The solubilized dECM digests, and/or their physically gelled hydrogels were characterized for their rheological properties, gelation kinetics, GAG content, proteomic composition, and growth factor profile. This study highlights papain as a plant-derived enzyme that can serve as a cost-effective alternative to animal-derived pepsin for the efficient solubilization of dECM. The resulting homogeneous papain-digested dECM preserved its thermally triggered gelation properties similar to pepsin digests, and the corresponding dECM hydrogels demonstrated their enhanced bioadhesiveness in single-cell force spectroscopy experiments with fibroblasts. The viability and proliferation of human HepaRG cells on dECM gels were similar to those on pure rat tail collagen type I gels. Papain is not only highly effective and economically attractive for dECM solubilization but also particularly interesting when digesting human-tissue-derived dECM for regenerative applications, where animal-derived materials are to be avoided.

The Ins and Outs of Clusterin: Its Role in Cancer, Eye Diseases and Wound Healing.

Clusterin (CLU) is a glycoprotein originally discovered in 1983 in ram testis fluid. Rapidly observed in other tissues, it was initially given various names based on its function in different tissues. In 1992, it was finally named CLU by consensus. Nearly omnipresent in human tissues, CLU is strongly expressed at fluid-tissue interfaces, including in the eye and in particular the cornea. Recent research has identified different forms of CLU, with the most prominent being a 75-80 kDa heterodimeric protein that is secreted. Another truncated version of CLU (55 kDa) is localized to the nucleus and exerts pro-apoptotic activities. CLU has been reported to be involved in various physiological processes such as sperm maturation, lipid transportation, complement inhibition and chaperone activity. CLU was also reported to exert important functions in tissue remodeling, cell-cell adhesion, cell-substratum interaction, cytoprotection, apoptotic cell death, cell proliferation and migration. Hence, this protein is sparking interest in tissue wound healing. Moreover, CLU gene expression is finely regulated by cytokines, growth factors and stress-inducing agents, leading to abnormally elevated levels of CLU in many states of cellular disturbance, including cancer and neurodegenerative conditions. In the eye, CLU expression has been reported as being severely increased in several pathologies, such as age-related macular degeneration and Fuch's corneal dystrophy, while it is depleted in others, such as pathologic keratinization. Nevertheless, the precise role of CLU in the development of ocular pathologies has yet to be deciphered. The question of whether CLU expression is influenced by these disorders or contributes to them remains open. In this article, we review the actual knowledge about CLU at both the protein and gene expression level in wound healing, and explore the possibility that CLU is a key factor in cancer and eye diseases. Understanding the expression and regulation of CLU could lead to the development of novel therapeutics for promoting wound healing.

Myeloid Plasticity of Neutrophil Precursors Contributes to Eosinophil Emergency Hematopoiesis

Abstract Eosinophils and neutrophils are heterogenous cells that play diverse roles in homeostasis and inflammation. Tissue eosinophil origins are poorly understood, posing challenges in the functional interpretation of heterogeneous phenotypes. New evidence from our lab shows unexpected hematopoietic plasticity of Ly6G(+)IL-5Rα(+) band-stage neutrophils with potential to contribute to eosinophil lineage. Using flow cytometry and multi-omics approaches in mouse and human tissue samples derived from healthy and allergic conditions, we tested the hypothesis that neutrophil plasticity contributes to eosinophil diversity in inflammation. We found at least two distinct populations of neutrophils at a healthy baseline, one of which expressed classical neutrophils markers (S100A8/9) while the other expressed both neutrophil (MPO, ELANE) and eosinophil-specific (EPX, PRG2) granule proteins, indicating an intermediate hematopoietic state. In human and mouse neutrophils treated with IL-5/G-CSF in vitro, a subset with mixed characteristics acquired eosinophil-specific granular proteins and eosinophil cell surface markers (Siglec8, CCR3). In a mouse asthma model, using scRNA-seq, we found overlap in neutrophils and eosinophils with tissue remodeling functions. We also identified activated neutrophils and eosinophils playing unique roles in inflammation. Our findings implicate eosinophil emergency hematopoiesis driven by neutrophil plasticity as an overlooked process driving persistence of tissue eosinophils in inflammation. Supported by grants from GSK ISS and NIH R01

The Effect of Titanium Surface Topography on Adherent Macrophage Integrin and Cytokine Expression

Immunomodulatory biomaterials have the potential to stimulate an immune response able to promote constructive and functional tissue remodeling, as opposed to persistent inflammation and scar tissue formation. This study examined the effects of titanium surface modification on integrin expression and concurrent cytokine secretion by adherent macrophages in vitro in an attempt to delineate the molecular events involved in biomaterial-mediated immunomodulation. Non-polarised (M0) and inflammatory polarised (M1) macrophages were cultured on a relatively smooth (machined) titanium surface and two proprietary modified rough titanium surfaces (blasted and fluoride-modified) for 24 h. The physiochemical characteristics of the titanium surfaces were assessed by microscopy and profilometry, while macrophage integrin expression and cytokine secretion were determined using PCR and ELISA, respectively. After 24 h adhesion onto titanium, integrin α1 expression was downregulated in both M0 and M1 cells on all titanium surfaces. Expression of integrins α2, αM, β1 and β2 increased in M0 cells cultured on the machined surface only, whereas in M1 cells, expression of integrins α2, αM and β1 all increased with culture on both the machined and rough titanium surfaces. These results correlated with a cytokine secretory response whereby levels of IL-1β, IL-31 and TNF-α increased significantly in M1 cells cultured on the titanium surfaces. These results show that adherent inflammatory macrophages interact with titanium in a surface-dependent manner such that increased levels of inflammatory cytokines IL-1β, TNF-α and IL-31 secreted by M1 cells were associated with higher expression of integrins α2, αM and β1.

Decellularized extracellular matrix (d-ECM): the key role of the inflammatory process in pre-regeneration after implantation.

Clinical medicine is encountering the challenge of repairing soft-tissue defects. Currently, natural and synthetic materials have been developed as natural scaffolds. Among them, the decellularized extracellular matrix (d-ECM) can achieve tissue remodeling following injury and, thus, replace defects due to its advantages of the extensiveness of the source and excellent biological and mechanical properties. However, by analyzing the existing decellularization techniques, we found that different preparation methods directly affect the residual components of the d-ECM, and further have different effects on inflammation and regeneration of soft tissues. Therefore, we analyzed the role of different residual components of the d-ECM after decellularization. Then, we explored the inflammatory process and immune cells in an attempt to understand the mechanisms and causes of tissue degeneration and regeneration after transplantation. In this paper, we summarize the current studies related to updated protocols for the preparation of the d-ECM, biogenic and exogenous residual substances, inflammation, and immune cells influencing the fate of the d-ECM.

Titanium Implant Surface Effects on Adherent Macrophage Phenotype: A Systematic Review.

Immunomodulatory biomaterials have the potential to stimulate an immune response able to promote constructive and functional tissue remodeling responses as opposed to persistent inflammation and scar tissue formation. As such, the controlled activation of macrophages and modulation of their phenotype through implant surface modification has emerged as a key therapeutic strategy. Methods: Online databases were searched for in vitro studies between January 1991 and June 2020 which examined the effect of titanium implant surface topography on the adherent macrophage phenotype at either the gene or protein level. Results: Thirty-nine studies were subsequently included for review. Although there was significant heterogeneity between studies, treatment of titanium surfaces increased the surface roughness or hydrophilicity, and hence increased macrophage attachment but decreased cell spreading. Physical coating of the titanium surface also tended to promote the formation of cell clusters. Titanium and titanium-zirconium alloy with a micro- or nano-scale rough topography combined with a hydrophilic surface chemistry were the most effective surfaces for inducing an anti-inflammatory phenotype in adherent macrophages, as indicated by significant changes in cytokine gene expression and or cytokine secretion profiles. Conclusions: The published data support the hypothesis that incorporation of specific topographical and physiochemical surface modifications to titanium can modulate the phenotypic response of adherent macrophages.

Assessing the response of human primary macrophages to defined fibrous architectures fabricated by melt electrowriting.

The dual role of macrophages in the healing process depends on macrophage ability to polarize into phenotypes that can propagate inflammation or exert anti-inflammatory and tissue-remodeling functions. Controlling scaffold geometry has been proposed as a strategy to influence macrophage behavior and favor the positive host response to implants. Here, we fabricated Polycaprolactone (PCL) scaffolds by Melt Electrowriting (MEW) to investigate the ability of scaffold architecture to modulate macrophage polarization.Primary human macrophages unpolarized (M0) or polarized into M1, M2a, and M2c phenotypes were cultured on PCL films and MEW scaffolds with pore geometries (square, triangle, and rhombus grid) characterized by different angles.M0, M2a, and M2c macrophages wrapped along the fibers, while M1 macrophages formed clusters with rounded cells. Cell bridges were formed only for angles up to 90°. No relevant differences were found among PCL films and 3D scaffolds in terms of surface markers. CD206 and CD163 were highly expressed by M2a and M2c macrophages, with M2a macrophages presenting also high levels of CD86. M1 macrophages expressed moderate levels of all markers. The rhombus architecture promoted an increased release by M2a macrophages of IL10, IL13, and sCD163 compared to PCL films. The proangiogenic factor IL18 was also upregulated by the rhombus configuration in M0 and M2a macrophages compared to PCL films.The interesting findings obtained for the rhombus architecture represent a starting point for the design of scaffolds able to modulate macrophage phenotype, prompting investigations addressed to verify their ability to facilitate the healing process in vivo.

Increased expression of prokineticin 2 and its receptor in endometrium of recurrent implantation failure patients decreased the expression of MMP9 important for decidualization

BackgroundStudies have shown that abnormalities in the decidualization process were closely related to recurrent implantation failure (RIF). Prokineticin 2 (PK2) is a secreted protein with angiogenic and tissue remodeling functions but its role in the endometrium is unknown.MethodsPK2 levels and its receptor PKR1 mRNA and protein levels in mid-secretory endometrium from normal and RIF women were examined by real-time PCR and western blotting, respectively. The effects of PK2 were evaluated by overexpressed PK2 in immortalized endometrial T-HESC cells using lentivirus vector and found different expression of Matrix metalloproteinase 9(MMP9) and lncRNA LUCAT1 by RNA-sequencing. The ability of PK2 to regulate LUCAT1 and MMP9 was verified in endometrial cells by real-time PCR and western blotting.ResultsUsing endometrial biopsies from normal and RIF patients, we found increased expression of PK2, together with its receptor PKR1 in RIF patients. We then overexpressed PK2 in immortalized endometrial T-HESC cells using lentivirus vector and found decreased expression of Matrix metalloproteinase 9(MMP9), and increased expression of lncRNA LUCAT1. We verified the ability of PK2 to stimulate LUCAT1 and decrease MMP9 in endometrial cells. We further demonstrated that increased expression of a long noncoding RNA LUCAT1 and decreased expression of MMP9 in endometrial biopsies of patients with RIF. Thus, we highlighted the important role of PK2 and its receptor PKR1 in decidualization and RIF.ConclusionProkineticin 2 and its receptor are important in endometrium decidualization. PK2 may affect endometrial decidualization through the LUCAT1- MMP9 pathway, thereby affecting embryo implantation.

Suppression of HIV-associated Macrophage Activation by a p75 Neurotrophin Receptor Ligand.

Previous studies indicated that nerve growth factor (NGF) and proNGF differentially regulate the phenotype of macrophages and microglia via actions at tropomyosin receptor kinase A (TrkA) and p75 neurotrophin receptors (p75NTR), respectively. The ability of HIV gp120 and virions to induce the secretion of factors toxic to neurons was suppressed by NGF and enhanced by proNGF, suggesting the potential for neurotrophin based "anti-inflammatory" interventions. To investigate the "anti-inflammatory" potential of the p75NTR ligand, LM11A-31, we treated cultured macrophages and microglia with HIV gp120 in the presence or absence of the ligand and evaluated the morphological phenotype, intrinsic calcium signaling, neurotoxic activity and proteins in the secretome. LM11A-31 at 10nM was able to suppress the release of neurotoxic factors from both monocyte-derived macrophages (MDM) and microglia. The protective effects correlated with a shift in morphology and a unique secretory phenotype rich in growth factors that overrode the actions of HIV gp120. The protein pattern was generally consistent with anti-inflammatory, phagocytic and tissue remodeling functions. Although the toxic factor(s) and the source of the neuroprotection were not identified, the data indicated that an increased degradation of NGF induced by HIV gp120 was likely to contribute to neuronal vulnerability. Although substantial work is still needed to reveal the functions of many proteins in the mononuclear phagocyte secretome, such as growth and differentiation factors, the data clearly indicate that the ligand LM11A-31 has excellent therapeutic potential due to its ability to induce a more protective phenotype that restricts activation by HIV.

Proinflammatory Oscillations Over the Menstrual Cycle Drives Bystander CD4 T Cell Recruitment and SHIV Susceptibility from Vaginal Challenge

Background: The menstrual cycle influences HIV infection-risk in women, although the timing and underlying mechanism are unclear. Here we investigated the contribution of the menstrual cycle to HIV susceptibility through evaluating immune behavior with infection-risk over time. Methods: Pigtail macaques were used to evaluate immune changes over reproductive cycles and in a low-dose repetitive vaginal virus challenge model. PBMC samples from healthy women were collected over the course of a cycle and measured for T cell characteristics. Immune properties from PBMC and vaginal lavage samples were measured by flow cytometry. Progesterone concentration from blood plasma was measured by enzyme immunoassay. The oscillation frequency of progesterone concentration and CCR5 expression from CD4 T cells was calculated using the Lomb-Scargle periodogram. SHIV infection was detected from blood plasma by Q-PCR. Findings: From macaques, cycle-phases associated with fluctuations in systemic immune properties which identified a type-1 inflammatory response bias with corresponding CCR5+ CD4 T cell (HIV target cell) infiltration into the vaginal lumen at the late luteal phase. Power spectral analysis detected CCR5 oscillation frequencies synchronized with reproductive cycles, and in a repetitive low-dose vaginal challenge model, productive SHIV163P3 infection occurred from inoculation during those intervals of mounting type-1 responses. Finally, we observe similar sinusoidal inflammatory properties in healthy menstruating women. Interpretation: These data demonstrate that periodic shifts in the immune landscape under menstrual cycle regulation drive bystander CCR5+ CD4 T cell recruitment, corresponding to the tissue remodeling functions at the late luteal phase, and increases HIV susceptibility in the female reproductive tract. Funding Statement: This study was supported by the U.S. Centers for Disease Control and Prevention, Atlanta, GA 30329 and NIH grants to Emory University (K23AI114407to A.N.S., the Emory University Center for AIDS research [P30AI050409], and Atlanta Clinical and Translational Sciences Institute [KLR2TR000455, UL1TR000454]). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: All [animal] procedures were approved by the Institutional CDC Animal Care and Use Committee (IACUC). Protocols were approved by the Emory University Institutional Review Board, the U.S. Centers for Disease Control and Prevention Institutional Review Board, and the Grady Research Oversight Committee.

Diverse homeostatic and immunomodulatory roles of immune cells in the developing mouse lung at single cell resolution.

At birth, the lungs rapidly transition from a pathogen-free, hypoxic environment to a pathogen-rich, rhythmically distended air-liquid interface. Although many studies have focused on the adult lung, the perinatal lung remains unexplored. Here, we present an atlas of the murine lung immune compartment during early postnatal development. We show that the late embryonic lung is dominated by specialized proliferative macrophages with a surprising physical interaction with the developing vasculature. These macrophages disappear after birth and are replaced by a dynamic mixture of macrophage subtypes, dendritic cells, granulocytes, and lymphocytes. Detailed characterization of macrophage diversity revealed an orchestration of distinct subpopulations across postnatal development to fill context-specific functions in tissue remodeling, angiogenesis, and immunity. These data both broaden the putative roles for immune cells in the developing lung and provide a framework for understanding how external insults alter immune cell phenotype during a period of rapid lung growth and heightened vulnerability.

Comparison of the host macrophage response to synthetic and biologic surgical meshes used for ventral hernia repair

The host innate immune response to a surgical mesh is arguably the most important determinant of tissue remodeling and functional outcome. Macrophage phenotype and the associated secretion of pro-inflammatory or anti-inflammatory cytokines during the first 10–14 days following implantation has been strongly associated with downstream events such as chronic inflammation vs. functional tissue remodeling, respectively, and the associated clinical consequences. A persistent, pro-inflammatory (M1-like) macrophage phenotype is typically associated with fibrosis and scarring. In contrast, an early transition to a regulatory, pro-remodeling (M2-like) macrophage phenotype is predictive of organized, site-appropriate connective tissue deposition. The ratio of M2-like to M1-like macrophages in the early post-implantation period defines the microenvironmental milieu and the associated tissue response. The present study evaluated the early macrophage response to a synthetic non-resorbable (Bard® Mesh), synthetic resorbable (TIGR® Matrix Surgical Mesh and GORE® BIO-A® Tissue Reinforcement), synthetic mesh composed of the naturally occurring molecule 4-hydroxybutyrate, (Phasix™ Mesh), and a biologic surgical mesh composed of dermal extracellular matrix (Strattice™ Reconstructive Tissue Matrix); all of which are used in ventral hernia repair. The spatiotemporal distribution of pro-inflammatory (CD68+CD86+TNF-α+) and pro-remodeling (CD68+CD206+) macrophages, and the remodeling response in terms of vascularization, total number of infiltrating cells, presence of multinucleate giant cells (MNGC), and cell layer thickness around the implanted materials was evaluated at 3, 7, 14, 21, and 35 days post implantation. Results showed an association of the synthetic non-resorbable and resorbable meshes with a robust, pro-inflammatory response within 3 days of implantation, and an increased presence of MNGC around the mesh fibers at longer time points. Phasix™ Mesh was associated with an increased presence of M2-like macrophages immediately adjacent to the mesh fibers at earlier time points, and a favorable tissue remodeling outcome at 35 days. Results of the present study are consistent with the premise that an early shift of M1-like to M2-like macrophages is associated with favorable outcomes, including reduced fibrosis, at later time points.

Wnt signaling regulates macula densa structure and function

The macula densa (MD), a plaque of 15–20 specialized cells, sits at the vascular pole of the glomerulus and plays a critical role in maintaining renal blood flow and glomerular filtration rate via tubuloglomerular feedback. Changes in renin state of the juxtaglomerular apparatus are known to be associated with alterations in MD cell morphology, although the underlying mechanism and importance of this phenomenon have been elusive. The canonical Wnt‐β catenin signaling pathway is known to regulate several cellular processes including embryonic development, cell proliferation, differentiation and homoeostasis. Glycogen synthase kinase 3 beta (GSK3b), a serine/threonine kinase, is involved in the phosphorylation and subsequent destablization of β catenin; thus, GSK3b plays a critical role in regulating the Wnt‐β catenin pathway. Lithium is a commonly used inhibitor of GSK3b in the kidney. Given the importance of Wnt‐β catenin in maintaining epithelial cell structure, the present studies aimed to explore its role in regulating MD morphology and function. Wild‐type mice were treated with lithium chloride for 2 weeks (40 mmol/kg chow for 7 days, and 60 mmol LiCl/kg chow subsequently), followed by harvesting and histological analysis of the kidneys. Lithium treatment caused a significant increase in the number of proliferating MD cells as quantified by Ki67 and EdU staining. As compared to control mice, MD cell number in the plaque was higher in lithium‐treated groups with several hyperplastic MD plaques observed containing > 40 cells. MD nucleus‐to‐cell volume ratio was 30% higher after lithium treatment. Using a O‐propargyl‐puromycin incorporation‐based protein synthesis imaging assay, a 3‐fold increase in protein synthesis was observed in MD cells after lithium treatment. Identical MD cell morphological alterations were observed in the newly generated nNOS‐Ctnnb1(Ex3) mice, a Wnt gain‐of‐function genetic model, confirming MD cell Wnt specificity of lithium treatment. In summary, Wnt signaling is an important regulator of MD morphology and cell biology, which may be involved in traditional renin and hemodynamic and novel tissue remodeling functions of MD cells.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Extracellular matrix hydrogels from decellularized tissues: Structure and function.

More than 70 papers have been published on extracellular matrix (ECM) hydrogels created from source tissue in almost every organ system. The present manuscript represents a review of ECM hydrogels and attempts to identify structure-function relationships that influence the tissue remodeling outcomes and gaps in the understanding thereof. There is a Phase 1 clinical trial now in progress for an ECM hydrogel.

Serum albumin as a local therapeutic agent in cell therapy and tissue engineering.

Albumin is a major plasma protein that has become ubiquitous in regenerative medicine research. As such, many studies have examined its structure and advantageous properties. However, a systematic and comprehensive understanding of albumin's role, capabilities and therapeutic potential still eludes the field. In the present work, we review how albumin is applied in tissue engineering, including cell culture and storage, in vitro fertilization and transplantation. Furthermore, we discuss how albumin's physiological role extends beyond a carrier for metal ions, fatty acids, pharmacons and growth factors. Albumin acts as a bacteriostatic coating that simultaneously promotes attachment and proliferation of eukaryotic cells. These properties with the combination of free radical scavenging, neutrophil activation and as a buffer molecule already make the albumin protein beneficial in healing processes supporting functional tissue remodeling. Nevertheless, recent data revealed that albumin can be synthesized by osteoblasts and its local concentration is raised after bone trauma. Interestingly, by increasing the local albumin concentration in vivo, faster bone healing is achieved, possibly because albumin recruits endogenous stem cells and promotes the growth of new bone. These data also suggest an active role of albumin, even though a specific receptor has not yet been identified. Together, this discussion sheds light on why the extravascular use of the albumin molecule is in the scope of scientific investigations and why it should be considered as a local therapeutic agent in regenerative medicine. © 2016 BioFactors, 43(3):315-330, 2017.

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry.

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

Immunomodulation and Mobilization of Progenitor Cells by Extracellular Matrix Bioscaffolds for Volumetric Muscle Loss Treatment.

Acellular bioscaffolds composed of extracellular matrix (ECM) have been effectively used to promote functional tissue remodeling in both preclinical and clinical studies of volumetric muscle loss, but the mechanisms that contribute to such outcomes are not fully understood. Thirty-two C57bl/6 mice were divided into eight groups of four animals each. A critical-sized defect was created in the quadriceps muscle and was repaired with a small intestinal submucosa ECM bioscaffold or left untreated. Animals were sacrificed at 3, 7, 14, or 56 days after surgery. The spatiotemporal cellular response in both treated and untreated groups was characterized by immunolabeling methods. Early time points showed a robust M2-like macrophage phenotype following ECM treatment in contrast to the predominant M1-like macrophage phenotype present in the untreated group. ECM implantation promoted perivascular stem cell mobilization, increased presence of neurogenic progenitor cells, and was associated with myotube formation. These cell types were present not only at the periphery of the defect near uninjured muscle, but also in the center of the ECM-filled defect. ECM bioscaffolds modify the default response to skeletal muscle injury, and provide a microenvironment conducive to a constructive healing response.

Liver immunology and its role in inflammation and homeostasis.

The human liver is usually perceived as a non-immunological organ engaged primarily in metabolic, nutrient storage and detoxification activities. However, we now know that the healthy liver is also a site of complex immunological activity mediated by a diverse immune cell repertoire as well as non-hematopoietic cell populations. In the non-diseased liver, metabolic and tissue remodeling functions require elements of inflammation. This inflammation, in combination with regular exposure to dietary and microbial products, creates the potential for excessive immune activation. In this complex microenvironment, the hepatic immune system tolerates harmless molecules while at the same time remaining alert to possible infectious agents, malignant cells or tissue damage. Upon appropriate immune activation to challenge by pathogens or tissue damage, mechanisms to resolve inflammation are essential to maintain liver homeostasis. Failure to clear ‘dangerous' stimuli or regulate appropriately activated immune mechanisms leads to pathological inflammation and disrupted tissue homeostasis characterized by the progressive development of fibrosis, cirrhosis and eventual liver failure. Hepatic inflammatory mechanisms therefore have a spectrum of roles in the healthy adult liver; they are essential to maintain tissue and organ homeostasis and, when dysregulated, are key drivers of the liver pathology associated with chronic infection, autoimmunity and malignancy. In this review, we explore the changing perception of inflammation and inflammatory mediators in normal liver homeostasis and propose targeting of liver-specific immune regulation pathways as a therapeutic approach to treat liver disease.