Regenerative medicine for end-stage fibrosis and tissue loss in the upper aerodigestive tract: a twenty-first century review.

OBJECTIVE: This review assesses regenerative medicine of the upper aerodigestive tract during the first two decades of the twenty-first century, focusing on end-stage fibrosis and tissue loss in the upper airways, salivary system, oropharynx and tongue. METHOD: PubMed, Embase, Google Scholar, Cochrane Library, Medline and clinicaltrials.org were searched from 2000 to 2019. The keywords used were: bioengineering, regenerative medicine, tissue engineering, cell therapy, regenerative surgery, upper aerodigestive tract, pharynx, oropharynx, larynx, trachea, vocal cord, tongue and salivary glands. Original studies were subcategorised by anatomical region. Original human reports were further analysed. Articles on periodontology, ear, nose and maxillofacial disorders, and cancer immunotherapy were excluded. RESULTS: Of 716 relevant publications, 471 were original studies. There were 18 human studies included, within which 8 reported airway replacements, 5 concerned vocal fold regeneration and 3 concerned salivary gland regeneration. Techniques included cell transplantation, injection of biofactors, bioscaffolding and bioengineered laryngeal structures. CONCLUSION: Moderate experimental success was identified in the restoration of upper airway, vocal fold and salivary gland function. This review suggests that a shift in regenerative medicine research focus is required toward pathology with a higher disease burden.

Linked chromosome 16q13 chemokines, macrophage-derived chemokine, fractalkine, and thymus- and activation-regulated chemokine, are expressed in human atherosclerotic lesions.

Chemokines are important mediators of macrophage and T-cell recruitment in a number of inflammatory pathologies, and chemokines expressed in atherosclerotic lesions may play an important role in mononuclear cell recruitment and macrophage differentiation. We have analyzed the expression of the linked chromosome 16q13 genes that encode macrophage-derived chemokine (MDC/CCL22), thymus- and activation-regulated chemokine (TARC/CCL17), and the CX(3)C chemokine fractalkine (CX(3)CL1) in primary macrophages and human atherosclerotic lesions by reverse transcription-polymerase chain reaction and immunohistochemistry. We show that macrophage expression of the chemokines MDC, fractalkine, and TARC is upregulated by treatment with the Th2-type cytokines interleukin-4 and interleukin-13. High levels of MDC, TARC, and fractalkine mRNA expression are seen in some, but not all, human arteries with advanced atherosclerotic lesions. Immunohistochemistry shows that MDC, fractalkine, and TARC are expressed by a subset of macrophages within regions of plaques that contain plaque microvessels. We conclude that MDC, fractalkine, and TARC, which are chromosome 16q13 chemokines, could play a role in mononuclear cell recruitment into atherosclerotic lesions and influence the subsequent inflammatory response. Macrophage-expressed chemokines upregulated by interleukin-4 may be useful surrogate markers for the presence of Th2-type immune responses in human atherosclerotic lesions.

The factor V Leiden (R506Q) mutation and risk of thrombosis in renal transplant recipients.

BACKGROUND: Renal transplantation and chronic renal failure are associated with an increased risk of venous thrombosis and myocardial infarction (MI). We investigated whether resistance to activated protein C due to a mutation in the factor V gene (FV Leiden/FV506Q) may predispose patients to thrombosis. METHODS: Three hundred patients who had undergone renal transplantation were genotyped for the FV mutation. Seventy-seven patients who had suffered thrombotic complications (42 venous, 28 arterial, and 7 both) were compared with 223 patients free of thrombosis. RESULTS: Thirty-two patients had suffered early renal allograft thrombosis (30 venous), and 33 patients had suffered MI. A higher proportion of the patients with thrombosis, compared to those without, had a family history of arterial cardiovascular disease (42% vs. 26%, P=0.04). Eighteen (6%) patients were heterozygous for FV506Q and seven (39%) of these had suffered venous thrombosis (including four primary allograft thromboses), compared with 15% of the patients without the mutation (P<0.05). The odds ratio for risk of venous thrombosis for FV506Q carriers was 3.6 (95% confidence interval: 1.3-9.9) or 4.0 (1.2-13.8) for primary allograft thrombosis. Only one of the FV506Q carriers had suffered an MI. CONCLUSIONS: Carriers of the factor V 506Q mutation with chronic renal failure who have undergone transplantation are at an increased risk of venous but not arterial thrombosis. This mutation explained 14% of all venous and 20% of primary allograft thrombosis, suggesting that other unidentified genetic and environmental factors contribute to the risk of thrombosis in renal transplant recipients.

Variation in the promoter region of the beta fibrinogen gene is associated with plasma fibrinogen levels in smokers and non-smokers.

We investigated the association between fibrinogen levels and a HaeIII restriction fragment length polymorphism located at -453 bp from the start of transcription of the beta fibrinogen gene. 292 healthy men aged 45 to 69 years, recruited from general practices throughout Britain, were studied. None had a history of ischaemic heart disease. 41.1% (120) were smokers and fibrinogen levels were higher in this group. The frequency of the non-cutting allele (designated H2) was 0.19 and was the same in smokers and non-smokers. The H2 allele was associated with elevated levels of fibrinogen in both smokers and non-smokers and the effect of genotype was similar in both groups. After smoking, HaeIII genotype was the strongest predictor of fibrinogen levels and explained 3.1% of the variance in fibrinogen levels. These results confirm earlier studies that variation at the fibrinogen locus contributes to the between-individual differences in plasma fibrinogen level.

The 4G/5G genetic polymorphism in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene is associated with differences in plasma PAI-1 activity but not with risk of myocardial infarction in the ECTIM study. Etude CasTemoins de I'nfarctus du Mycocarde.

We have investigated the interrelationships of plasma PAI-1 activity, the PAI-1 4G/5G polymorphism and risk of myocardial infarction (MI) in the ECTIM study, a case-control study of MI based in Belfast, Lille, Strasbourg and Toulouse. Mean PAI-1 levels in cases were similar across all centres but in controls, levels in the French centres were significantly higher. Only in Belfast were PAIl1 levels higher in cases (11.7 AU/ml) than controls (10.5 AU/ml). The PAI-1 4G allele frequency was similar in cases and controls (0.55 and 0.54). In all groups, 4G homozygotes had the highest mean plasma PAI-1 level (4G4G vs 5G5G; cases overall: 14.2 vs 12.1AU/ml; controls overall: 15.0 vs 12.6AU/ml), with the heterozygotes generally intermediate. The data from Belfast are consistent with the literature implicating PAI-1 level as an MI risk factor. In ECTIM, the PAI-1 4G/5G polymorphism is not a genetic risk factor for MI but is associated with PAI-1 activity. Thus homozygosity for the 4G allele may predispose to elevated PAI-1 and impaired fibrinolysis, perhaps requiring interaction with other genetic or environmental factors to influence MI risk.

Genetic factors determining thrombosis and fibrinolysis.

Raised plasma levels of fibrinogen, factor VIIc, and plasminogen activator inhibitor-1 (PAI-1) are associated with an increased risk of ischemic heart disease. Levels of these proteins are determined in part by environmental influences such as smoking and dietary fat intake. However, genetic variation explains much of the interindividual variation in plasma levels of these proteins not accounted for by environmental factors. We previously investigated the DNA variation at the fibrinogen gene locus and showed that BclI restriction fragment length polymorphism (RFLP) of the beta-fibrinogen gene is associated with between-person differences in plasma fibrinogen levels. This RFLP is unlikely to be the functional base change itself, since it lies downstream of the gene. The rate-limiting step in the production of the mature fibrinogen molecule in the human hepatoma cell-line HepG2 is the synthesis of the beta-polypeptide chain, which in turn is influenced by the amount of messenger (mRNA) available. One possibility is that BclI RFLP is in linkage disequilibrium with a base change in the region of the beta-gene controlling synthesis of its mRNA and ultimately of fibrinogen protein. We identified a base change in the 5'-flanking region of the beta-fibrinogen gene that is in linkage disequilibrium with the BclI RFLP, that is associated with plasma fibrinogen levels, and that may be involved in control of fibrinogen gene expression. For the factor VII gene, we identified a polymorphism, detected after Msp I digestion of polymerase chain reaction (PCR)-amplified genomic DNA, that is strongly associated with factor VII coagulant activity (factor VIIc). The base change that creates the Msp I polymorphism is a G to A substitution, leading to the replacement of arginine (Arg) with glutamine (Gln) in the protein product of the M2 allele. In a sample of 284 men from the United Kingdom the frequency of the Gln allele (M2 loss of cutting site) is 0.1, and individuals of genotype Arg/Gln have factor VIIc levels 22% below the sample mean. In this sample, the Msp I genotype was found to be the strongest predictor of factor VIIc, accounting for 20.2% of the variance, with cholesterol accounting for an additional 3.5%. Three individuals homozygous for the Gln allele had both low factor VIIc and low factor VII protein concentrations. The conformation of the factor VII Gln may be different from that of the Arg protein, affecting its intracellular processing, secretion, turnover in plasma, or activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Fibrinogen polymorphisms and atherothrombotic disease.

Common polymorphisms of the fibrinogen gene cluster are associated with circulating fibrinogen level and with susceptibility to and/or severity of atherothrombotic disease. The frequencies of the polymorphisms vary among different ethnic groups but there is strong linkage disequilibrium at the beta-fibrinogen gene locus so that, in caucasian populations, there are only four common beta-fibrinogen haplotypes. One of these haplotypes, defined by the beta-fibrinogen -455A allele, is associated with elevated fibrinogen level and increased risk of atherothrombotic disease. The molecular mechanism of these associations is currently under investigation.

The study of gene-environment interactions that influence thrombosis and fibrinolysis. Genetic variation at the loci for factor VII and plasminogen activator inhibitor-1.

We describe studies on variation in the genes coding for factor VII (FVII) and plasminogen activator inhibitor-1 (PAI-1) that influence levels of these proteins in the blood. For FVII, we have identified a genetic polymorphism that results in the substitution of arginine at residue 353 to glutamine. The frequency of the glutamine allele is approximately 0.1 in samples of individuals from the United Kingdom (n = 777) and the United States (n = 140) and in Afro-Caribbeans (n = 49), and is significantly higher in a sample of individuals from the Indian subcontinent (n = 53). In all samples, carriers of the glutamine allele had levels of FVII coagulant activity 20% to 25% lower than those with only the arginine allele. These differences were highly statistically significant in the United Kingdom sample. This effect was consistent in healthy men and women and in those with coronary artery disease. In individuals homozygous for the glutamine allele, both FVII coagulant activity and antigen are low, and the mechanism of the association appears likely to be due to an effect on secretion from the liver or stability in the plasma. In individuals in the general population FVII coagulant activity is positively correlated with levels of plasma triglycerides, due to the effect of such lipoproteins on activation of FVII. This relationship appears weaker in individuals carrying the glutamine allele, and since elevated FVII coagulant antibody is associated with risk of thrombosis, this is an example of how environmental factors may interact with an individual's genotype to determine his or her thrombotic risk. Roughly 20% of the general population are carriers of the glutamine allele and are likely to be genetically protected from such risk. For PAI-1, we have recently shown that variation at the PAI gene locus, detected as DNA polymorphisms, is associated with between-individual differences in levels of PAI-1. We have now detected a common sequence change in the promoter region of the gene that explains part of this effect. The sequence change is at position 675, where a fifth guanine (5G allele) has been inserted into a run of four guanines (4G allele) when compared with published sequences. In a sample of both 83 healthy individuals and 105 young patients with coronary artery disease from Sweden, the frequency of the 4G allele is roughly 0.5, and those individuals homozygous for the 4G allele have higher levels of PAI-1 than those with other genotypes (29% higher). Preliminary data show that the 5G allele binds a hepatic nuclear protein that the 4G allele does not, suggesting that the mechanism of the effect may be due to a direct effect of the sequence change on transcription of the PAI-1 gene.(ABSTRACT TRUNCATED AT 400 WORDS)

Environmental and genetic determinants of the hypercoagulable state and cardiovascular disease in renal transplant recipients.

BACKGROUND: Fibrinogen and factor VII coagulant activity (VIIc), risk factors for cardiovascular disease (CVD) in the general population, could contribute to CVD risk in renal transplant recipients (RTR). METHODS: We measured fibrinogen and VIIc in 38 RTR and 31 controls, along with prothrombin fragment F1 + 2 and D-Dimer (markers of coagulation and fibrinolytic activation), plasma lipids and the acute phase response cytokine, interleukin 6. The effect of genetic polymorphisms of beta-fibrinogen (G/A-455) and factor VII (Arg/Gln353) was explored. RESULTS: F1 + 2, D-Dimer, and fibrinogen were increased in all RTR, indicating a chronic prothrombotic state. Fibrinogen correlated with age. F1 + 2, and trough cyclosporin A (CsA). RTR carriers of the A-455 allele had a greater increment in plasma fibrinogen concentration and correlation with CsA than homozygotes for the G-455 allele. Interleukin 6 was increased in RTR confirming that a persistent lowgrade acute-phase response could contribute to increased fibrinogen. Differences in plasma VIIc were associated with factor VII genotype, disease status, and blood lipids. Carriers of the Gln353 allele had 30% lower VIIc when compared with Arg353 homozygotes, which could confer a reduced thrombotic risk. The 12 RTR with CVD or metabolic complications (RTR+) were more hyperlipidaemic and had higher fibrinogen and VIIc than the 26 RTR free of disease complications (RTR-), or the controls. CONCLUSIONS: Long-term RTR manifest features of a chronic prothrombotic and persistent inflammatory state. Alterations in fibrinogen and VIIc in RTR arise in part as a result of interactions between common genetic and environmental factors, and these changes could contribute to the increased risk of CVD in RTR.

Factor VII coagulant activity (VIIc) and hypercoagulability in chronic renal disease and dialysis: relationship with dyslipidaemia, inflammation, and factor VII genotype.

BACKGROUND: Factor VII coagulant activity (VIIc) is implicated in cardiovascular disease (CVD) risk in the general population. VIIc is correlated with hyperlipidaemia and influenced by a polymorphism of the factor VII gene and could contribute to thrombotic risk in patients with renal disease. METHODS: We studied VIIc in 100 patients with chronic renal disease or on maintenance dialysis and examined its relationship with dyslipidaemia, a marker of coagulation activation prothrombin fragment F1+2 (F1+2), the acute-phase reactant and coagulation factor fibrinogen, a mediator of the inflammatory response interleukin-6 (IL6), and the factor VII R353Q polymorphism. RESULTS: VIIc (186+/-58 vs 140+/-37, % standard, P<0.0001) and F1+2 (0.51 vs 0.30 nM, median, P<0.0001) were increased in the patients with renal disease compared with the control group, consistent with a hypercoagulable state. Patients and controls heterozygous for the factor VII R353Q polymorphism, had 35% lower VIIc than homozygotes for the R353 allele, indicating that the Q353 allele could confer genetic protection from thrombotic risk. There was a significant correlation between VIIc and F1+2 (r=0.26, P<0.05), total and VLDL cholesterol, and triglycerides, but the correlation with lipids did not differ by genotype. VIIc and F1+2 also correlated with increased concentration of IL6 and fibrinogen, and inversely with albumin, suggesting that a persistent inflammatory response could contribute to a hypercoagulable state, possibly via cytokine induced activation of the endothelium, or by induction of monocytes to express tissue factor. Patients with CVD complications or a history of myocardial infarction did not have higher VIIc or F1+2 than those without CVD. CONCLUSIONS: VIIc was significantly increased in renal disease states and strongly influenced by a common polymorphism of the factor VII gene, but the increase in VIIc and its correlation with lipids was not genotype specific. VIIc correlated with evidence of increased coagulation activation and persistence of an inflammatory response. A persistent inflammatory response and the dyslipidaemia of renal disease may contribute to coagulation activation and increased cardiovascular risk. Prospective studies are required to evaluate increased VIIc as a thrombotic risk factor in chronic renal disease.

Biological and physical properties of a model calcitonin containing a glutamate residue interrupting the hydrophobic face of the idealized amphiphilic alpha-helical region.

2A new calcitonin analogue, model calcitonin III (MCt-III), has been synthesized, and its biological and physical characteristics have been studied. This analogue has an idealized alpha-helix from residue 8-22 with glutamate at position 15 interrupting an otherwise continuous surface of aliphatic side chains (those of leucine residues) on the hydrophobic face of the helix. MCt-III differs from a previous model, MCt-II, only by the substitution Leu15----Glu and is here compared with salmon calcitonin I (sCt-I) and MCt-II to elucidate further the role of the putative amphiphilic alpha-helix in determining biological and physical properties of the hormone. MCt-III shows physical properties intermediate between those of sCt-I and MCt-II, demonstrating the influence of appropriately positioned single residues on properties of amphiphilic structures. In our two biological assays, a brain-binding assay and an in vivo hypocalcemic assay, MCt-III reproduces the sigmoidal dose-response curves of sCt-I; this contrasts with the behavior of MCt-II, which demonstrated unusual dose-response curves in these two assays. MCt-III is almost three times more potent than sCt-I in our hypocalcemic assay; this activity groups MCt-III among the most potent known analogues of sCt-I.

Cooperative influence of genetic polymorphisms on interleukin 6 transcriptional regulation.

Interleukin 6 (IL6) plays key roles in hematopoiesis, immune, and acute phase responses. Dysregulated IL6 expression is implicated in diseases such as atherosclerosis and arthritis. We have examined the functional effect of four polymorphisms in the IL6 promoter (-597G-->A, -572G-->C, -373A(n)T(n), -174G-->C) by identifying the naturally occurring haplotypes and comparing their effects on reporter gene expression. The results indicate different transcriptional regulation in the ECV304 cell line compared with the HeLa cell line, suggesting cell type-specific regulation of IL6 expression. The haplotypes showed functional differences in the ECV304 cell line; transcription was higher from the GG9/11G haplotype and lower from the AG8/12G allele. The differences suggest that more than one of the polymorphic sites is functional; the base differences at distinct polymorphic sites do not act independently of one another, and one polymorphism influences the functional effect of variation at other polymorphic sites. These results show that genetic polymorphisms in the promoter influence IL6 transcription not by a simple additive mechanism but rather through complex interactions determined by the haplotype.

Association of genetic variation at the beta-fibrinogen gene locus and plasma fibrinogen levels; interaction between allele frequency of the G/A-455 polymorphism, age and smoking.

The beta-fibrinogen G/A-455 polymorphism genotype along with plasma fibrinogen levels was determined in 482 healthy middle-aged men, of whom 231 were smokers. Smokers had the highest plasma fibrinogen levels (2.92 g/l), ex-smokers the next (2.73 g/l), and never-smokers the lowest levels (2.66 g/l, P < 0.001). Those with one or two A-455 alleles had significantly higher plasma fibrinogen levels in never-smokers and ex-smokers (8.2% and 9.0%, respectively, P < 0.05), and the effect was larger in younger men (45 < 55 years, 11.6%, P = 0.002) than older men (> 65 years, 4.5%, NS), and was not significant in smokers (2.4%, P > 0.05). Allele frequencies were calculated and compared across age groups and between smokers and non-smokers. The difference in frequency of the A-455 allele between smokers and non-smokers varied significantly with age (P < 0.01), with the frequency of the A-455 allele being significantly lower in smokers than in non-smokers in subjects aged > 65 years (P < 0.05), but not in younger men. This demonstrates an interaction between age, smoking and allele frequency of the G/A-455 polymorphism in this population-based sample.