Organ Cultures Of Tissue Research Articles

Clinical outcome of organ‐cultured preloaded DMEK compared to standard DMEK ‐ A single center study

The use of ready‐to‐use grafts from specialized eye banks might provide a number of benefits, including graft quality control, assured availability at a certain operation time, a decreased likelihood of case cancellation, and a shorter and less sophisticated DMEK surgery, with a resulting lower risk of graft damage. However, it is critical to thoroughly establish the clinical safety of employing these preloaded tissues. Especially since most of the studies were prepared and stored under hypothermic conditions. There are only a few studies on preprepared tissues in organ culture, which are partly controversial.In this prospective study we included patients who received DMEK surgery at the Knappschaft Eye Clinic Sulzbach between 05/2021–12/2022. Patients received either a preloaded DMEK (pDMEK), prepared 5 days before surgery in the eye bank, or a conventional, directly before surgery, surgeon‐prepared DMEK (sDMEK).The preliminary data shows that preloaded DMEK is a safe procedure, but there is a trend towards more frequent rebubbling in the pDMEK group and a statistically non‐significant lower postoperative endothelial cell count compared to the sDMEK group. However, visual acuity and corneal thickness reduction were similar in both groups.

Human immature testicular tissue organ culture: a step towards fertility preservation and restoration.

Cryopreservation of immature testicular tissue (ITT) is currently the only option to preserve fertility of prepubertal patients. Autologous transplantation of ITT may not be safe or appropriate for all patients. Therefore, methods to mature ITT ex vivo are needed. Aim to investigate the feasibility of inducing in vitro spermatogenesis from ITT cryopreserved for pediatric patients prior to initiation of gonadotoxic therapy. Cryopreserved-thawed ITT from prepubertal and peripubertal patients were cultured for 7, 16, and 32 days in medium with no hormones or supplemented with 5 IU/L FSH, 1 IU/L hCG, or 5IU/L FSH+1 IU/L hCG. Samples were evaluated histologically to assess tissue integrity, and immunofluorescence staining was performed to identify VASA (DDX4)+ germ cells, UCHL1+ spermatogonia, SYCP3+ spermatocytes, CREM+ spermatids, SOX9+ Sertoli cells. Proliferation (KI67) and apoptosis (CASPASE3) of germ cells and Sertoli cells were also analyzed. Sertoli and Leydig cell maturation was evaluated by AR and INSL3 expression as well as expression of the blood testis barrier protein, CLAUDIN11, and testosterone secretion in the culture medium. Integrity of seminiferous tubules, VASA+ germ cells and SOX9+ Sertoli cells were maintained up to 32 days. The number of VASA+ germ cells was consistently higher in the peripubertal groups. UCHL1+ undifferentiated spermatogonia and SOX9+ Sertoli cell proliferation was confirmed in most samples. SYCP3+ primary spermatocytes began to appear by day 16 in both age groups. Sertoli cell maturation was demonstrated by AR expression but the expression of CLAUDIN11 was disorganized. Presence of mature and functional Leydig cells was verified by INSL3 expression and secretion of testosterone. Gonadotropin treatments did not consistently impact the number or proliferation of germ cells or somatic cells, but FSH was necessary to increase testosterone secretion over time in prepubertal samples. ITT were maintained in organotypic culture for up to 32 days and spermatogonia differentiated to produce primary spermatocytes in both pre- and peripubertal age groups. However, complete spermatogenesis was not observed in either group.

CXCR4 and CXCL12 signaling regulates the development of extrinsic innervation to the colorectum.

The gastrointestinal tract is innervated by an intrinsic neuronal network, known as the enteric nervous system (ENS), and by extrinsic axons arising from peripheral ganglia. The nerve of Remak (NoR) is an avian-specific sacral neural crest-derived ganglionated structure that extends from the cloaca to the proximal midgut and, similar to the pelvic plexus, provides extrinsic innervation to the distal intestine. The molecular mechanisms controlling extrinsic nerve fiber growth into the gut is unknown. In vertebrates, CXCR4, a cell-surface receptor for the CXCL12 chemokine, regulates migration of neural crest cells and axon pathfinding. We have employed chimeric tissue recombinations and organ culture assays to study the role of CXCR4 and CXCL12 molecules in the development of colorectal innervation. CXCR4 is specifically expressed in nerve fibers arising from the NoR and pelvic plexus, while CXCL12 is localized to the hindgut mesenchyme and enteric ganglia. Overexpression of CXCL12 results in significantly enhanced axonal projections to the gut from the NoR, while CXCR4 inhibition disrupts nerve fiber extension, supporting a previously unreported role for CXCR4 and CXCL12 signaling in extrinsic innervation of the colorectum.

Soluble CD13 induces inflammatory arthritis by activating the bradykinin receptor B1.

CD13, an ectoenzyme on myeloid and stromal cells, also circulates as a shed, soluble protein (sCD13) with powerful chemoattractant, angiogenic, and arthritogenic properties, which require engagement of a G protein–coupled receptor (GPCR). Here we identify the GPCR that mediates sCD13 arthritogenic actions as the bradykinin receptor B1 (B1R). Immunofluorescence and immunoblotting verified high expression of B1R in rheumatoid arthritis (RA) synovial tissue and fibroblast-like synoviocytes (FLSs), and demonstrated binding of sCD13 to B1R. Chemotaxis, and phosphorylation of Erk1/2, induced by sCD13, were inhibited by B1R antagonists. In ex vivo RA synovial tissue organ cultures, a B1R antagonist reduced secretion of inflammatory cytokines. Several mouse arthritis models, including serum transfer, antigen-induced, and local innate immune stimulation arthritis models, were attenuated in Cd13–/– and B1R–/– mice and were alleviated by B1R antagonism. These results establish a CD13/B1R axis in the pathogenesis of inflammatory arthritis and identify B1R as a compelling therapeutic target in RA and potentially other inflammatory diseases.

Long-term preservation of human donor corneal tissues in organ culture.

Long-term preservation of human donor corneal tissues in organ culture.

Regulation of adipose tissue lipolysis by ghrelin is impaired with high-fat diet feeding and is not restored with exercise

ABSTRACT Ghrelin is released from the stomach as an anticipatory signal prior to a meal and decreases immediately after. Previous research has shown that both acylated (AG) and unacylated (UnAG) ghrelin blunt adrenoreceptor-stimulated lipolysis in rat white adipose tissue (WAT) ex vivo. We investigated whether acute or chronic consumption of a high fat diet (HFD) impaired the ability of ghrelin to regulate adipose tissue lipolysis, and if this impairment could be restored with exercise. After 5 days (5d) of a HFD, or 6 weeks (6 w) of a HFD (60% kcal from fat) with or without exercise training, inguinal and retroperitoneal WAT was collected from anesthetized rats for adipose tissue organ culture. Samples were treated with 1 μM CL 316,243 (CL; lipolytic control), 1 μM CL+150 ng/ml AG or 1 μM CL+150 ng/ml UnAG. Incubation media and tissue were collected after 2 hours. Colorometric assays were used to determine glycerol and free fatty acid (FFA) concentrations in media. Western blots were used to quantify the protein content of lipolytic enzymes and ghrelin receptors in both depots. CL stimulated lipolysis was evidenced by increases in glycerol (p < 0.0001) and FFA (p < 0.0001) concentrations in media compared to control. AG decreased CL-stimulated glycerol release in inguinal WAT from 5d LFD rats (p = 0.0097). Neither AG nor UnAG blunted lipolysis in adipose tissue from 5d or 6 w HFD-fed rats, and exercise did not restore ghrelin’s anti-lipolytic ability in 6 w HFD-fed rats. Overall, this study demonstrates that HFD consumption impairs ghrelin’s ability to regulate adipose tissue lipolysis.

Human Nasal Turbinate Tissues in Organ Culture as a Model for Human Cytomegalovirus Infection at the Mucosal Entry Site.

The initial events of viral infection at the primary mucosal entry site following horizontal person-to-person transmission have remained ill defined. Our limited understanding is further underscored by the absence of animal models in the case of human-restricted viruses, such as human cytomegalovirus (HCMV), a leading cause of congenital infection and a major pathogen in immunocompromised individuals. Here, we established a novel ex vivo model of HCMV infection in native human nasal turbinate tissues. Nasal turbinate tissue viability and physiological functionality were preserved for at least 7 days in culture. We found that nasal mucosal tissues were susceptible to HCMV infection, with predominant infection of ciliated respiratory epithelial cells. A limited viral spread was demonstrated, involving mainly stromal and vascular endothelial cells within the tissue. Importantly, functional antiviral and proleukocyte chemotactic signaling pathways were significantly upregulated in the nasal mucosa in response to infection. Conversely, HCMV downregulated the expression of nasal epithelial cell-related genes. We further revealed tissue-specific innate immune response patterns to HCMV, comparing infected human nasal mucosal and placental tissues, representing the viral entry and the maternal-to-fetal transmission sites, respectively. Taken together, our studies provide insights into the earliest stages of HCMV infection. Studies in this model could help evaluate new interventions against the horizontal transmission of HCMV.IMPORTANCE HCMV is a ubiquitous human pathogen causing neurodevelopmental disabilities in congenitally infected children and severe disease in immunocompromised patients. The earliest stages of HCMV infection in the human host have remained elusive in the absence of a model for the viral entry site. Here, we describe the establishment and use of a novel nasal turbinate organ culture to study the initial steps of viral infection and the consequent innate immune responses within the natural complexity and the full cellular repertoire of human nasal mucosal tissues. This model can be applied to examine new antiviral interventions against the horizontal transmission of HCMV and potentially that of other viruses.

Analysis of Factors Influencing the Suitability of Donor Corneas in the LIONS Cornea Bank Saar-Lor-Lux, Trier/Westpfalz from 2006 to 2016

With the discrepancy between availability and demand for corneal donors, the purpose of our study was to identify significant factors influencing the suitability and affecting the main reasons for discarding organ-cultured corneas. We thus aimed to minimise the percentage of unsuitable corneas in the long term. 4140 corneas of 2083 donors were analysed between 2006 and 2016. The factors included age, gender and medical history of the donors as well as storage factors such as post-mortem times. The data was recruited in a Microsoft Access database and evaluated with SPSS 20.0, using logistic regression analyses. In a multiple regression model, six factors influencing the suitability of the donor tissue were analysed. All corneas that did not meet the criteria for elective keratoplasty were considered unsuitable. A high donor age of > 80 years was found to be a significantly negative factor on the suitability of the cornea (p = 0.001) compared to the reference group of donors < 40years. Previous cataract surgery (p < 0.001) also significantly reduced suitability. Male corneas were significantly less suitable than female corneas (p = 0.028). Hypertension (p = 0.612), chemotherapy (p = 0.745) or diabetes mellitus (p = 0.321) did not significantly affect the suitability. Sepsis (p = 0.026) and multiorgan failure (p < 0.001) as cause of death resulted in significantly higher discarding rates due to contamination of the culture medium. Corneas in which the blood sample collection took more than 12 h from death of the donor had to be discarded significantly more frequently due to a positive donor serology. Factors such as a history of cataract surgery and high donor age reduced the suitability of corneal donor tissue in organ culture. Diabetes, arterial hypertension and chemotherapy in the donor history did not statistically significantly affect the suitability of donor tissue. Causes of death such as sepsis or multi-organ failure increased the contamination rate of the medium. Post-mortem blood collection after more than 12 h should be avoided due to increased positive serology results.

In Vitro and Ex Vivo Models for Functional Testing of Therapeutic Anti-scarring Drug Targets in Keloids.

Significance: Keloids are benign fibro-proliferative raised dermal lesions that spread beyond the original borders of the wound, continue to grow, rarely regress, and are the most common in pigmented individuals after an abnormal wound healing response. The current treatment failure and respective challenges involved highlighting the underlying issue that the etiopathogenesis of keloids is still not well understood. Disease models are required to better understand the disease pathogenesis. It is not possible to establish keloids in animals because of the uniqueness of this disease to human skin. To address this challenge, along these lines, non-animal reproducible models are vital in investigating molecular mechanisms of keloid pathogenesis and therapeutics development. Recent Advances: Various non-animal models have been developed to better understand the molecular mechanisms involved in keloid scarring and aid in identifying and evaluating the therapeutic potential of novel drug candidates. In this scenario, the current review aims at describing in vitro monocultures, co-cultures, organotypic cultures, and ex vivo whole skin keloid tissue organ culture models. Critical Issues and Future Directions: Current treatment options for keloids are far from securing a cure or preventing disease recurrence. Identifying universally accepted effective therapy for keloids has been hampered by the absence of appropriate disease model systems. Animal models do not accurately mimic the disease, thus non-animal model systems are pivotal in keloid research. The use of these models is essential not only for a better understanding of disease biology but also for identifying and evaluating novel drug targets.

Multi-dimensional models for functional testing of keloid scars: In silico, in vitro, organoid, organotypic, ex vivo organ culture, and in vivo models.

Keloid scars are described as benign fibro-proliferative dermal outgrowths that commonly occur in pigmented skin post cutaneous injury, and continue to grow beyond the boundary of the original wound margin. There is a lack of thorough understanding of keloid pathogenesis and thus keloid therapeutic options remain ill-defined. In view of the poor response to current therapy and high recurrence rates, there is an unmet need in improving our knowledge and therefore in identifying targeted and effective treatment strategies in management of keloids. Keloid research however, is hampered by a lack of relevant animal models as keloids do not spontaneously occur in animals and are unique to human skin. Therefore, developing novel animal models and nonanimal models for functional evaluation of keloid cells and tissue for better understanding their pathobiology and response to putative candidate therapies are essential. Here, we present the key concepts and relevant emerging research on two-dimensional and three-dimensional cell and tissue models for functional testing of keloid scars. We will describe in detail current models including in vitro mono- and co-cultures, multi-cellular spheroids (organoids) and organotyopic cultures, ex vivo whole skin keloid tissue organ culture models as well as in vivo human patient models. Finally, we discuss the role played by time as the fourth dimension in a novel model that involves sequential temporal biopsies of human patients with keloids (a so called 4D in vivo human model). The use of these unique models will no doubt prove pivotal in identification of new drug targets as well as biomarkers, in functional testing of emerging novel therapeutics, and in enhancing our understanding of keloid disease biology.

The Pathogenic Neisseria Use a Streamlined Set of Peptidoglycan Degradation Proteins for Peptidoglycan Remodeling, Recycling, and Toxic Fragment Release.

Neisseria gonorrhoeae and Neisseria meningitidis release peptidoglycan (PG) fragments from the cell as the bacteria grow. For N. gonorrhoeae these PG fragments are known to cause damage to human Fallopian tube tissue in organ culture that mimics the damage seen in patients with pelvic inflammatory disease. N. meningitidis also releases pro-inflammatory PG fragments, but in smaller amounts than those from N. gonorrhoeae. It is not yet known if PG fragment release contributes to the highly inflammatory conditions of meningitis and meningococcemia caused by N. meningitidis. Examination of the mechanisms of PG degradation and recycling identified proteins required for these processes. In comparison to the model organism E. coli, the pathogenic Neisseria have far fewer PG degradation proteins, and some of these proteins show differences in subcellular localization compared to their E. coli homologs. In particular, some N. gonorrhoeae PG degradation proteins were demonstrated to be in the outer membrane while their homologs in E. coli were found free in the periplasm or in the cytoplasm. The localization of two of these proteins was demonstrated to affect PG fragment release. Another major factor for PG fragment release is the allele of ampG. Gonococcal AmpG was found to be slightly defective compared to related PG fragment permeases, thus leading to increased release of PG. A number of additional PG-related factors affect other virulence functions in Neisseria. Endopeptidases and carboxypeptidases were found to be required for type IV pilus production and resistance to hydrogen peroxide. Also, deacetylation of PG was required for virulence of N. meningitidis as well as normal cell size. Overall, we describe the processes involved in PG degradation and recycling and how certain characteristics of these proteins influence the interactions of these pathogens with their host.

Serum-Free Organ Culture of the Embryonic Mouse Ureter.

The ability to explant and then maintain embryonic tissues in organ culture makes it feasible to study the growth and differentiation of whole organs, or parts or combinations of them, in three dimensions. Moreover, the possible effects of biochemical manipulations or mutations can be explored by visualizing a growing organ. The mammalian renal tract comprises the kidney, ureter, and urinary bladder, and the focus of this chapter is organ culture of the embryonic mouse ureter in serum-free defined medium. Over the culture period, rudiments grow in length, smooth muscle differentiates, and the ureters then undergo peristalsis in a proximal to distal direction.

Cloning of the Coffea canephora SERK1 promoter and its molecular analysis during the cell-to-embryo transition

Somatic embryogenesis receptor-like kinase 1 (SERK1) is a cell membrane receptor active in different plant tissues and involved in cell differentiation activities including somatic embryogenesis. The identification of promoter elements responsible for SERK1 expression during the onset of somatic embryogenesis can be useful to understand the molecular regulation of the cell-to embryo transition, and these promoter elements represent biotechnological tools in plant organ tissue culture. A − 1,620 bp DNA sequence located upstream of the Coffea canephora SERK1 gene homologue (CcSERK1) was isolated, and then, different segments containing key response elements (REs) for somatic embryogenesis onset and development were fused to the uidA (encoding a β-glucuronidase, GUS) reporter gene to evaluate its expression in transgenic leaf explants. DNA segments of − 1,620 and − 1048 bp in length directed uidA expression with patterns in leaf explants similar to those occurring during somatic embryogenesis. When a − 792-bp fragment was used, uidA expression disappeared only in leaf explants and pro-embryogenic mass but persisted in developing embryos. No uidA expression was detected in any embryogenic stage when a − 618-bp fragment was used. DNA deletions showed that a − 1048-bp sequence located upstream of the CcSERK1 gene is sufficient to direct gene expression during the onset and the development of C. canephora somatic embryogenesis. The DNA segment located between − 1048 and − 792 bp (containing BBM and WUS REs) is needed for gene expression before embryogenesis onset but not during embryo development. The promoter segment between − 792 and − 618 bp (including GATA, ARR1AT, and ANT REs) regulates gene expression in developing embryos. How to cite: Jiménez-Guillen D, Pérez-Pascual D, Souza-Perera R, et al. Cloning of the Coffea canephora SERK1 promoter and its molecular analysis during the cell-to-embryo transition. Electron J Biotechnol 2018;36.https://doi.org/10.1016/j.ejbt.2018.08.005.

Ghrelin Blunts Adrenergic‐Stimulated Lipolysis in Subcutaneous and Visceral Adipose Tissue Ex Vivo, but not In Vivo

IntroductionGhrelin is an appetite and growth hormone (GH) ‐stimulating gastric hormone. Ghrelin rises preprandially and immediately declines to baseline after meal consumption. Therefore, it is important to elucidate any potential role that ghrelin may have in mediating substrate metabolism surrounding entrained meal time. There is evidence to suggest that ghrelin may inhibit the adrenergic stimulation of lipolysis in isolated adipocytes, which would seem intuitive if we consider ghrelin as a meal‐priming signal, as there is unlikely to be a requirement to spare blood glucose following a meal. However, upon administration of ghrelin in humans, there are marked increases in local adipose and skeletal muscle rates of glycerol appearance, indicative of increased lipolysis. In vivo findings, though, are confounded by a secondary increase in GH, which may be lipolytic. Previously, using an ex vivo (adipose tissue organ culture) model, we have shown that both acylated (AG) and unacylated (UnAG) ghrelin blunt adrenergic (CL 316 243) stimulated lipolysis, with a corresponding reduction in the activation of hormone‐sensitive lipase (HSL). Our current experimental model is aimed at elucidating whether or not this newly found, direct role for ghrelin in mediating adipose tissue lipolysis can be replicated in vivo.MethodsTo date, subcutaneous iWAT and visceral RP adipose tissue depots have been harvested from healthy, male Sprague‐Dawley rats for the assessment of glycerol release following injection with either saline, CL (1mg/kg), CL + AG and CL + UnAG (50μg/kg). In vivo tissue and blood collection was done 30min following IP injection. Western blots were used to quantify the activation of markers associated with lipolysis. qPCR will be undertaken to assess markers of fatty acid storage (eg. fatty acid synthase). ELISA will be used to quantify GH, insulin and glucagon as potential circulating confounders.ResultsEx vivo, AG and UnAG blunted CL‐stimulated lipolysis, but did not independently affect glycerol release or lipolytic signalling proteins (p&gt;0.05) and were not pursued further with in vivo injection studies. When compared to saline (0.36 ± 0.06 mM), CL injection markedly increased the rates of glycerol release (0.77 ± 0.07 mM; p&lt;0.05). There was no effect of co‐administration of CL with ghrelin isoforms on the CL‐mediated increase in glycerol release (AG: 0.77 ± 0.04; UnAG: 0.79 ± 0.04 mM). These outcomes were mirrored by signalling proteins, in that HSL (Serine563/660) activation was significantly elevated with CL (iWAT: 2.80 ± 0.43; RP: 4.69 ± 1.23) compared to saline injection (iWAT: 0.78 ± 0.23; RP: 2.13 ± 1.03) and unchanged with the co‐injection ghrelin (iWAT ‐ CL+AG: 2.61 ± 0.54, CL+UnAG: 2.39 ± 0.22; RP – CL+AG: 6.83 ± 1.89, CL+UnAG: 11.66 ± 2.93).ConclusionsWe extend on previous findings and show that ghrelin directly inhibits adrenergic‐stimulated lipolysis in subcutaneous and visceral adipose depots. However, in the living animal, these actions appear to be confounded by other factors. Transcriptional markers of lipid storage will be used to elucidate whether ghrelin is also acting directly as a storage signal, beyond its inhibition of fatty acid mobilization. These experiments will be seminal in contributing to the interpretation of AG and UnAG's effects in adipose tissue lipid metabolism.Support or Funding InformationFunded by NSERCThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In Vitro Modeling of Co-Transplantation of Multipotent Stromal Mesenchymal Cells from Orbital Fat Pad and Lipoaspirate of Human Subcutaneous Adipose Tissue in Organ Culture in Collagen Gel.

The interplay of multipotent stromal cells derived from the orbital fat pads and cells of the lipoaspirate from the subcutaneous adipose tissue was studied using in vitro co-transplantation model in an organ culture in a collagen gel. Microscopy findings and intensity of apoptosis and cell proliferation in cultures of lipoaspirate with and without multipotent stromal cells showed that the cells maintained their viability, proliferation capacity, and cytokine secretion activity. Higher proliferatitive activity of cells in cocultures promotes renewal of fat transplant cells and can help to maintain its stable volume in delayed terms after transplantation.

Estradiol does not directly regulate adipose lipolysis

ABSTRACTThe mechanisms by which estradiol modulates adipose lipolysis are poorly understood. We sought to measure basal and β3-stimulated indices of lipoysis (FFAs, glycerol) in vivo in E2 deficient or supplemented rats, and ex vivo with direct acute E2 exposure. For 2 weeks, ovariectomized (OVX) and OVX rats treated with a daily oral dose of E2 (OVX E2) were pairfed to SHAM controls (n = 12 per group). Adipocyte size was modestly (∼40%) increased in OVX rats, but did not reach significance (p = 0.2). After 2 weeks, half of the animals in each group received an in vivo injection of saline or 1 mg/kg of the β3 agonist CL 316, 243. Serum FFA concentrations, but not glycerol, were lower in OVX and OVX E2 rats compared with SHAM controls (p = 0.02). A significant CL response was present in all groups (p<0.001) and HSL activation was unaffected by OVX or OVX E2 in retroperitoneal (r.p.) or inguinal (iWAT) adipose depots in vivo. Ex vivo, CL increased FFA and glycerol accumulation in the media as well as HSL phosphorylation by several fold in r.p. and iWAT explants, but responses from OVX and OVX E2 rats were comparable to SHAMs. To assess whether E2 can directly affect lipolysis, r.p. and iWAT tissue was treated with E2, CL or E2 + CL for 2, 4 or 8 hours using adipose tissue organ culture. CL stimulated FFA release (p<0.001), but was unaffected by E2. Overall, our results indicate that E2 does not directly regulate adipose tissue lipolysis.

Atrogin-1 Increases Smooth Muscle Contractility Through Myocardin Degradation.

Atrogin-1, an E3 ligase present in skeletal, cardiac and smooth muscle, down-regulates myocardin protein during skeletal muscle differentiation. Myocardin, the master regulator of smooth muscle cell (SMC) differentiation, induces expression of smooth muscle marker genes through its association with serum response factor (SRF), which binds to the CArG box in the promoter. Myocardin undergoes ubiquitylation and proteasomal degradation. Evidence suggests that proteasomal degradation of myocardin is critical for myocardin to exert its transcriptional activity, but there is no report about the E3 ligase responsible for myocardin ubiquitylation and subsequent transactivation. Here, we showed that overexpression of atrogin-1 increased contractility of cultured SMCs and mouse aortic tissues in organ culture. Overexpression of dominant-negative myocardin attenuated the increase in SMC contractility induced by atrogin-1. Atrogin-1 overexpression increased expression of the SM contractile markers while downregulated expression of myocardin protein but not mRNA. Atrogin-1 also ubiquitylated myocardin for proteasomal degradation in vascular SMCs. Deletion studies showed that atrogin-1 directly interacted with myocardin through its amino acids 284-345. Immunostaining studies showed nuclear localization of atrogin-1, myocardin, and the Rpt6 subunit of the 26S proteasome. Atrogin-1 overexpression not only resulted in degradation of myocardin but also increased recruitment of RNA Polymerase II onto the promoters of myocardin target genes. In summary, our results have revealed the roles for atrogin-1 in the regulation of smooth muscle contractility through enhancement of myocardin ubiquitylation/degradation and its transcriptional activity. J. Cell. Physiol. 232: 806-817, 2017. © 2016 Wiley Periodicals, Inc.

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface.

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm(3) pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

Distinct Adipose Depots from Mice Differentially Respond to a High-Fat, High-Salt Diet

Background:Dietary factors such as high-sodium or high-fat (HF) diets have been shown to induce a proinflammatory phenotype. However, there is limited information with respect to how microenvironments of distinct intra-abdominal adipose depots respond to the combination of a high-salt, HF diet.Objective:We tested the hypothesis that HF feeding would cause changes in distinct adipose depots, which would be further amplified by the addition of high salt to the diet.Methods:Twenty-seven male C57BL6 mice were fed an HF diet (60% of kcal from fat), an HF + high-salt diet (4% wt:wt), a control diet [low-fat (LF);10% of kcal from fat], or an LF + high-salt diet for 12 wk. The main sources of fat in the diets were corn oil and lard. Adipokines in serum and released from adipose tissue organ cultures were measured by immunoassays. QIAGEN's Ingenuity Pathway Analysis was used to perform functional analysis of the RNA-sequencing data from distinct adipose depots.Results:Diet-induced obesity resulted in a classical inflammatory phenotype characterized by increased concentrations of circulating inflammatory mediators (38–56%) and reduced adiponectin concentrations (27%). However, high-salt feeding did not exacerbate the HF diet–induced changes in adipokines and cytokines. Leptin and interleukin-6 were differentially released from adipose depots and HF feeding impaired adiponectin and resistin secretion across all 3 depots (34–48% and 45–83%, respectively). The addition of high salt to the HF diet did not further modulate secretion in cultured adipose tissue experiments. Although gene expression data from RNA sequencing indicated a >4.3-fold upregulation of integrin αX (Itgax) with HF feeding in all 3 depots, markers of cellular function were differentially expressed in response to diet across depots.Conclusion:Collectively, these findings highlight the role of distinct adipose depots in mice in the development of obesity and emphasize the importance of selecting specific depots to study the effects of therapeutic interventions on adipose tissue function.