Recent Advances in Liver Tissue Engineering as an Alternative and Complementary Approach for Liver Transplantation.

Acute and chronic liver diseases cause significant morbidity and mortality worldwide, affecting millions of people. Liver transplantation is the primary intervention method, replacing a non-functional liver with a functional one. However, the field of liver transplantation faces challenges such as donor shortage, postoperative complications, immune rejection, and ethical problems. Consequently, there is an urgent need for alternative therapies that can complement traditional transplantation or serve as an alternative method. In this review, we explore the potential of liver tissue engineering as a supplementary approach to liver transplantation, offering benefits to patients with severe liver dysfunctions.

Human acetyl-CoA:glucosamine-6-phosphate N-acetyltransferase 1 has a relaxed donor specificity and transfers acyl groups up to four carbons in length.

Glucosamine-6-phosphate N-acetyltransferase1 (GNA1) catalyses the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to glucosamine-6-phosphate (GlcN6P) to form N-acetylglucosamine-6-phosphate (GlcNAc6P), which is an essential intermediate in UDP-GlcNAc biosynthesis. An analog of GlcNAc, N-butyrylglucosamine (GlcNBu) has shown healing properties for bone and articular cartilage in animal models of arthritis. The goal of this work was to examine whether GNA1 has the ability to transfer a butyryl group from butyryl-CoA to GlcN6P to form GlcNBu6P, which can then be converted to GlcNBu. We developed fluorescent and radioactive assays and examined the donor specificity of human GNA1. Acetyl, propionyl, n-butyryl, and isobutyryl groups were all transferred to GlcN6P, but isovaleryl-CoA and decanoyl-CoA did not serve as donor substrates. Site-specific mutants were produced to examine the role of amino acids potentially affecting the size and properties of the AcCoA binding pocket. All of the wild type and mutant enzymes showed activities of both acetyl and butyryl transfer and can therefore be used for the enzymatic synthesis of GlcNBu for biomedical applications.

The use of marine-derived bioactive compounds as potential hepatoprotective agents.

The marine environment may be explored as a rich source for novel drugs. A number of marine-derived compounds have been isolated and identified, and their therapeutic effects and pharmacological profiles are characterized. In the present review, we highlight the recent studies using marine compounds as potential hepatoprotective agents for the treatment of liver fibrotic diseases and discuss the proposed mechanisms of their activities. In addition, we discuss the significance of similar studies in Oman, where the rich marine life provides a potential for the isolation of novel natural, bioactive products that display therapeutic effects on liver diseases.

Inflammatory cytokines promote growth of intestinal smooth muscle cells by induced expression of PDGF-Rß.

Thickening of the inflamed intestinal wall involves growth of smooth muscle cells (SMC), which contributes to stricture formation. Earlier, the growth factor platelet-derived growth factor (PDGF)-BB was identified as a key mitogen for SMC from the rat colon (CSMC), and CSMC growth in colitis was associated with both appearance of its receptor, PDGF-Rß and modulation of phenotype. Here, we examined the role of inflammatory cytokines in inducing and modulating the growth response to PDGF-BB. CSMC were enzymatically isolated from Sprague-Dawley rats, and the effect of tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, transforming growth factor (TGF), IL-17A and IL-2 on CSMC growth and responsiveness to PDGF-BB were assessed using proliferation assays, PCR and western blotting. Conditioned medium (CM) was obtained at 48 hrs of trinitrobenzene sulphonic acid-induced colitis. Neither CM alone nor cytokines caused proliferation of early-passage CSMC. However, CM from inflamed, but not control colon significantly promoted the effect of PDGF-BB. IL-1ß, TNF-α and IL-17A, but not other cytokines, increased the effect of PDGF-BB because of up-regulation of mRNA and protein for PDGF-Rß without change in receptor phosphorylation. PDGF-BB was identified in adult rat serum (RS) and RS-induced CSMC proliferation was inhibited by imatinib, suggesting that blood-derived PDGF-BB is a local mitogen in vivo. In freshly isolated CSMC, CM from the inflamed colon as well as IL-1ß and TNF-α induced the early expression of PDGF-Rß, while imatinib blocked subsequent RS-induced cell proliferation. Thus, pro-inflammatory cytokines both initiate and maintain a growth response in CSMC via PDGF-Rß and serum-derived PDGF-BB, and control of PDGF-Rß expression may be beneficial in chronic intestinal inflammation.

Impaired platelet-derived growth factor receptor expression and function in cultured lower esophageal sphincter circular smooth muscle cells from W/W(v) mutant mice.

We have previously demonstrated that lower esophageal sphincter (LES) circular smooth muscle (CSM) is functionally impaired in W/W(v) mutant mice that lack interstitial cells of Cajal, and speculated that this could be due to altered smooth muscle differentiation. Platelet-derived growth factor (PDGF) is involved in the maturation and differentiation of smooth muscle. To determine whether PDGF expression and (or) function is altered in W/W(v) mutant mice, PDGF-Rß expression was measured using RT-PCR, qPCR, and immunocytochemistry, and Ca(2+) imaging and perforated patch clamp recordings performed in isolated LES CSM cells. RT-PCR and immunocytochemistry showed significantly reduced PDGF-Rß expression in the LES from mutant as opposed to wild-type mice. Quantitative comparison of CSM cell numbers in histological specimens revealed a significantly increased average cell size in the mutant tissue. The specific PDGF-Rß ligand, PDGF-BB, caused a significant increase in intracellular Ca(2+) in cells from the wild-type mice compared with the mutants. Using a ramp protocol, PDGF-BB caused a 2-fold increase in outward K(+) currents in cells from the wild-type mice, whereas no significant increase was measured in the cells from the mutants. We conclude that the expression and function of PDGF-Rß in LES CSM from W/W(v) mice is impaired, providing further evidence that LES CSM is abnormal in W/W(v) mutants.

The pro-inflammatory cytokines IL-1ß and TNFα are neurotrophic for enteric neurons.

Intestinal inflammation causes initial axonal degeneration and neuronal death but subsequent axon outgrowth from surviving neurons restores innervation density to the target smooth muscle cells. Elsewhere, the pro-inflammatory cytokines TNFα and IL-1ß cause neurotoxicity, leading us to test their role in promoting enteric neuron death. In a rat coculture model, TNFα or IL-1ß did not affect neuron number but did promote significant neurite outgrowth to twofold that of control by 48 h, while other cytokines (e.g., IL-4, TGFß) were without effect. TNFα or IL-1ß activated the NFκB signaling pathway, and inhibition of NFκB signaling blocked the stimulation of neurite growth. However, nuclear translocation of NFκB in smooth muscle cells but not in adjacent neurons suggested a dominant role for smooth muscle cells. TNFα or IL-1ß sharply increased both mRNA and protein for GDNF, while the neurotrophic effects of TNFα or IL-1ß were blocked by the RET-receptor blocker vandetanib. Conditioned medium from cytokine-treated smooth muscle cells mimicked the neurotrophic effect, inferring that TNFα and IL-1ß promote neurite growth through NFκB-dependent induction of glial cell line-derived neurotrophic factor (GDNF) expression in intestinal smooth muscle cells. In vivo, TNBS-colitis caused early nuclear translocation of NFκB in smooth muscle cells. Conditioned medium from the intact smooth muscle of the inflamed colon caused a 2.5-fold increase in neurite number in cocultures, while Western blotting showed a substantial increase in GDNF protein. Pro-inflammatory cytokines promote neurite growth through upregulation of GDNF, a novel process that may facilitate re-innervation of smooth muscle cells and a return to homeostasis following initial damage.

Proliferation modulates intestinal smooth muscle phenotype in vitro and in colitis in vivo.

Intestinal inflammation causes an increased intestinal wall thickness, in part, due to the proliferation of smooth muscle cells, which impairs the contractile phenotype elsewhere. To study this, cells from the circular muscle layer of the rat colon (CSMC) were isolated and studied, both in primary culture and after extended passage, using quantitative PCR, Western blot analysis, and immunocytochemistry. By 4 days in vitro, both mRNA and protein for the smooth muscle marker proteins α-smooth muscle actin, desmin, and SM22-α were reduced by >50%, and mRNA for cyclin D1 was increased threefold, evidence for modulation to a proliferative phenotype. Continued growth caused significant further decrease in expression, evidence that phenotypic loss in CSMC was proportional to the extent of proliferation. In CSMC isolated at day 2 of trinitrobenzene sulfonic acid-induced colitis, flow cytometry and Western blotting showed that these differentiated markers were reduced in mitotic CSMC, while similar to control in nonmitotic CSMC. By day 35 post-trinitrobenzene sulfonic acid, when inflammation has resolved, CSMC were hypertrophic, but, nonetheless, showed markedly decreased expression of smooth muscle protein markers per cell. In vitro, day 35 CSMC displayed an accelerated loss of phenotype and increased thymidine uptake in response to serum or PDGF-BB. Furthermore, carbachol-induced expression of phospho-AKT (a marker of cholinergic response) was lost from day 35 CSMC in vitro, while retained in control cells. Therefore, proliferation reduces the expression of smooth-muscle-specific markers in CSMC, possibly leading to altered contractility. However, inflammation-induced proliferation in vivo also causes lasting changes that include unexpected priming for an exaggerated response to proliferative stimuli. Identification of the molecular mechanisms of intestinal smooth muscle cell phenotypic modulation will be helpful in reducing the detrimental effects of inflammation.

Mitogenic factors promoting intestinal smooth muscle cell proliferation.

Intestinal smooth muscle cells are normally quiescent, but in the widely studied model of trinitrobenzene sulfonic acid (TNBS)-induced colitis in the rat, the onset of inflammation causes proliferation that leads to increased cell number and an altered phenotype. The factors that drive this are unclear and were studied in primary cultures of circular smooth muscle cells (CSMC) from the rat colon. While platelet-derived growth factor (PDGF)-AA, fibroblast growth factor (FGF), and epidermal growth factor (EGF) were ineffective, PDGF-BB and insulin-like growth factor-1 (IGF-1) caused significant increase in [(3)H]thymidine incorporation, bromodeoxyuridine uptake, and increased CSMC number, with PDGF-BB (≥0.2 nM) substantially more effective than IGF-1. Surprisingly, CSMC lacked expression of PDGF receptor-ß (PDGF-Rß) upon isolation but by 4 days in vitro, CSMC gained expression of PDGF-Rß as shown by quantitative PCR, Western blot analysis, and immunocytochemistry; these CSMC responded to PDGF-BB but not IGF-1. PDGF-BB caused PDGF-Rß phosphorylation and mobilization from the surface membrane, leading to activation of both Akt and ERK signaling pathways, which were essential for subsequent proliferation. In contrast, PDGF-AA, FGF, EGF, and IGF-1 were ineffective. In vivo, control CSMC lacked expression of PDGF-Rß. However, this changed rapidly with TNBS-colitis, and by day 2 when CSMC proliferation in vivo is maximal, freshly isolated CSMC showed on-going PDGF-Rß phosphorylation that was further increased by exogenous PDGF-BB. This suggests that the onset of PDGF-Rß expression is a key factor in CSMC growth in vitro and in vivo, where inflammation may damage intrinsic inhibitory mechanisms and thus lead to hyperplasia.

Neuronal nitric oxide inhibits intestinal smooth muscle growth.

Hyperplasia of smooth muscle contributes to the thickening of the intestinal wall that is characteristic of inflammation, but the mechanisms of growth control are unknown. Nitric oxide (NO) from enteric neurons expressing neuronal NO synthase (nNOS) might normally inhibit intestinal smooth muscle cell (ISMC) growth, and this was tested in vitro. In ISMC from the circular smooth muscle of the adult rat colon, chemical NO donors inhibited [(3)H]thymidine uptake in response to FCS, reducing this to baseline without toxicity. This effect was inhibited by the guanylyl cyclase inhibitor ODQ and potentiated by the phosphodiesterase-5 inhibitor zaprinast. Inhibition was mimicked by 8-bromo (8-Br)-cGMP, and ELISA measurements showed increased levels of cGMP but not cAMP in response to sodium nitroprusside. However, 8-Br-cAMP and cilostamide also showed inhibitory actions, suggesting an additional role for cAMP. Via a coculture model of ISMC and myenteric neurons, immunocytochemistry and image analysis showed that innervation reduced bromodeoxyuridine uptake by ISMC. Specific blockers of nNOS (7-NI, NAAN) significantly increased [(3)H]thymidine uptake in response to a standard stimulus, showing that nNOS activity normally inhibits ISMC growth. In vivo, nNOS axon number was reduced threefold by day 1 of trinitrobenzene sulfonic acid-induced rat colitis, preceding the hyperplasia of ISMC described earlier in this model. We conclude that NO can inhibit ISMC growth primarily via a cGMP-dependent mechanism. Functional evidence that NO derived from nNOS causes inhibition of ISMC growth in vitro predicts that the loss of nNOS expression in colitis contributes to ISMC hyperplasia in vivo.

A cell-based assay for screening lipoxygenase inhibitors.

Lipoxygenases (LOX) form a family of lipid peroxidizing enzymes within the plant and animal kingdoms. In humans, six functional lipoxygenase isoforms have been identified. 5-LOX, "platelet-type" 12-LOX (p12-LOX) and 15-LOX type 1 (15-LOX1), originally identified in leukocytes, platelets, and reticulocytes, respectively, generate lipid mediators involved in host cellular functions and in the pathophysiology of asthma, cardiovascular diseases, and cancer. The pharmaceutical industry has reinvigorated their programs to develop novel LOX inhibitors in view of recent findings. However, high throughput LOX screening assays to test novel agents against these intracellular enzymes are limited. We describe a cell-based 96-well microplate fluorescence assay tested against several existing LOX inhibitors, and validate the assay by comparing known IC(50) values and HPLC analysis, which may provide a useful screen for novel LOX inhibitors.

Antimicrobial activity of omwaprin, a new member of the waprin family of snake venom proteins.

We have isolated and characterized omwaprin, a 50-amino-acid cationic protein from the venom of inland taipan (Oxyuranus microlepidotus). It is a new member of the waprin family of snake venom proteins. A synthetic gene was designed and constructed for expressing the recombinant protein in Escherichia coli. Recombinant omwaprin was used for carrying out functional analyses. The protein is non-toxic to Swiss albino mice at doses of up to 10 mg/kg when administered intraperitoneally. However, it shows selective and dose-dependant antibacterial activity against Gram-positive bacteria. The minimum inhibitory doses were in the range 2-10 microg for selected species of bacteria in radial diffusion assays. The antibacterial activity is salt-tolerant up to 350 mM NaCl. However, omwaprin lost its antibacterial activity upon reduction and alkylation of its cysteine residues, or upon deletion of six N-terminal amino acid residues, four of which are positively charged. These observations indicate that the three-dimensional structure constrained by four disulfide bonds and the N-terminal residues are essential for its activity. The mechanism of action is via membrane disruption, as shown by scanning electron microscopy. Importantly, omwaprin lacks haemolytic activity on human erythrocytes. This demonstrates the specificity of omwaprin for bacterial membranes. Unlike other reported WAP (whey acidic protein) domain-containing antibacterial proteins, including elafin, EPPIN (epididymal proteinase inhibitor), SWAM1 and SWAM2 [single WAP (whey acidic protein) motif proteins 1 and 2] and SLPI (secretory leucocyte proteinase inhibitor), omwaprin shows species-specific activity on the Gram-positive bacteria tested.