Figure 5 - uploaded by Manuela M. Santos
Content may be subject to copyright.
Heart fibrosis in  2mRag1 Ϫ / Ϫ mice kept on a standard diet (azan staining). a: The hearts of B6 control mice show a normal histology. b: Extensive fibrotic lesions (blue) are present in the heart of a 5-month-old  2mRag1 Ϫ / Ϫ mouse. c and d: Heart histology in radiation chimeras; animals were 2 months old at the start of the experiment. c: Prevention of heart fibrosis in a  2mRag1 Ϫ / Ϫ mouse reconstituted with fetal liver cells from B6 donor mice (9 months old). d:
Source publication
Genetic causes of hereditary hemochromatosis (HH) include mutations in the HFE gene, a ss2-microglobulin (ss2m)-associated major histocompatibility complex class I-like protein. Accordingly, mutant ss2m(-/-) mice have increased intestinal iron absorption and develop parenchymal iron overload in the liver. In humans, other genetic and environmental...
Contexts in source publication
Context 1
... Ϫ / Ϫ mice to Ͼ 80%, reaching levels similar to those seen in  2m - and  2mRag1 double-knockout mice kept on a standard diet. Plasma iron concentration in iron-loaded animals was significantly lower in B6 control mice compared to all of the other strains (B6: plasma iron Ͻ 29 versus Ͼ 50 mol Fe/ml in all other strains; P Ͻ 0.01). Taken together, these results show that iron burden is accentuated in dietary iron-loaded  2mRag1 double- knockout mice when compared to the respective single knockout mice. A typical feature of pathological iron overload in humans is the cellular distribution of storage iron, which has been particularly difficult to mimic in rodents. Therefore, we determined histologically the cellular distribution of storage iron in liver, pancreas, and heart in mice fed a standard diet and in dietary iron-loaded animals. Perl’s blue-staining of liver sections from  2m -single and  2mRag1 double-knockout mice kept on a standard diet revealed the presence of excess iron, which was predominantly in parenchymal cells (data not shown). 4,5 Moderate deposits were also observed in the pancreas and the heart of 24- to 30-week-old  2mRag1 double- knockout mice, but not in the  2m -single and Rag1 single-knockout mice or B6 wild-type mice (data not shown). As previously reported for shorter loading periods, iron deposition in the liver of B6 wild-type mice fed an iron-enriched diet up to 12 weeks was particularly prominent in Kupffer cells, and was also present in parenchymal cells (Figure 2a). Surprisingly, Rag1 single-knockout mice, that supposedly have normal Kupffer cells, develop hepatic iron overload on dietary iron loading exclusively in parenchymal cells (data not shown), like HH patients and  2m Ϫ / Ϫ mice. 5 Dietary iron-loaded  2mRag1 double-knockout mice show heavy iron depositions in the livers that corresponded to the appearance of hepato- cyte clusters (Figure 2b). A remarkable iron loading was present in the pancreas and the heart of  2mRag1 double-knockout mice (Figure 2, d and f), which was not observed in control B6 (Figure 2, c and e), and  2m single-knockout mice (data not shown). Importantly, in the pancreas this prominent iron deposition was present in acinar cells (Figure 2d), and in the heart it was present in myocytes and in the interstitial tissue (Figure 2f). Examination of hearts from  2mRag1 double-knockout mice by electron microscopy revealed frequent lysosomal structures containing granular electron-dense mate- rial in the cytoplasm of myocytes (Figure 3, a and b). Similar lysosomal iron deposition was observed in mes- enchymal perivascular cells. In the pancreas, the acinar cells contained large lysosomes of moderate electron density (Figure 3c). In these lysosomes, scattered ferritin particles were present (Figure 3d). Ferritin accumulation was also evident in the cytoplasm of acinar cells. Overall, dietary iron-loaded  2mRag1 double-knockout mice develop a more severe iron burden in multiple organs than each of the single-knockout mice, indicating an additive effect of the two mutations. To exclude the possibility that anemia could account for the abnormal iron storage defect in  2mRag1 double- knockout mice, several erythroid parameters were determined. The results demonstrated that hemoglobin, hematocrit, and mean corpuscular volume were even higher in  2m -single and in  2mRag1 double-knockout mice when compared to B6 and Rag1 Ϫ / Ϫ mice fed a standard diet (Table 2). We observed an increase of hemoglobin, hematocrit, and mean corpuscular volume values to a similar extent when B6 and Rag1 Ϫ / Ϫ mice were fed the iron-enriched diet for 12 weeks. Thus, the excess storage iron found in  2mRag1 double-knockout mice could not be attributed to defective erythropoiesis or hemoglobin synthesis. To investigate the effect of the Rag1 mutation on the absorption of iron, ferric iron, Fe(III), absorption 6 was measured before and after feeding an iron-enriched diet for 14 days (Figure 4). Ferric iron absorption after this treatment significantly decreased in all mouse strains ( P Ͻ 0.0001). However, iron absorption in  2m -single and  2mRag1 double-knockout mice was persistently higher, before and after treatment, when compared to wild-type (B6) or Rag1 single-knockout mice ( P Ͻ 0.0001, Figure 4). No significant differences were found between iron absorption in  2m -single and  2mRag1 double- knockout mice, indicating that the Rag1 mutation has no further influence on iron absorption in the gut. Iron deposition in the heart deserves special interest, because heart failure is a frequent cause of death in untreated HH and posttransfusional secondary hemochromatosis. 12–16 Remarkably, 17 out of 21  2mRag1 double-knockout mice aged between 20 and 28 weeks and kept on a standard diet developed heart fibrosis, as detected by azan staining, which was never seen in  2m and Rag1 -single-knockout mice or control mice of the same age and kept on the standard diet (Figure 5, a and b). Only after feeding an iron-enriched diet for 3 months, heart fibrosis was additionally observed in Rag1 single- knockout mice, but not in  2m single-knockout or B6 wild-type mice (data not shown). Previously we have demonstrated that reconstitution of  2m Ϫ / Ϫ mice with normal hematopoietic cells, redistrib- utes the iron from parenchymal to Kupffer cells in the liver. 5 To further investigate the influence of hematopoietic cells in the development of iron-related heart fibrosis, we reconstituted lethally irradiated 8-week-old  2mRag1 double-knockout mice with fetal liver-derived hematopoietic progenitor cells from normal mice. All reconstituted  2mRag1 double-knockout mice ( n ϭ 4) showed a normal histology up to 36 weeks of age (Figure 5 c ). The control  2mRag1 double-knockout mice reconstituted with  2mRag1 Ϫ / Ϫ -derived cells ( n ϭ 5) were sacrificed between 20 to 28 weeks when they became ill and had developed extensive fibrosis in the heart (Figure 5d). Thus, wild-type hematopoietic cell transfer prevents the development of heart fibrosis in  2mRag1 double-knockout mice. The aim of this study was to investigate the modifying influence of lymphocytes in the pathology of iron overload. Such a modifying role has been suggested by the association between low numbers of T lymphocytes in patients with HH and a more severe clinical expression of iron overload. 8 –10 In the  2m -deficient mice that develop a progressive iron overload similar to that seen in HH patients, 4 –7 we introduced the Rag1 mutation, 11 to create a total absence of mature lymphocytes. When kept on a standard diet, the double-knockout mice develop a more severe phenotype than the  2m deficient mice, involving increased iron accumulation in the liver, heart, and pancreas. The  2mRag1 double- knockout mice have visible iron depositions specifically in parenchymal cells of the liver and significantly higher iron levels in the heart than single-knockout and control mice. This indicates that the additional absence of lymphocytes, in the  2m model of iron overload, exacerbates the accumulation of iron in target organs, especially the heart. Moreover, the double-deficient mice spontane- ously develop fibrosis in the heart. The observed phenotype in the double-deficient mice is also an accentuation of the phenotype of the Rag1 single-knockout mice, which can normally regulate iron absorption and storage, and do not develop heart fibrosis under standard conditions. Rag1 single-knockout mice will develop heart fibrosis after very long periods of dietary iron loading of at least 12 weeks. Thus, dietary iron loading in combination with the lack of lymphocytes leads to cardiomyopathy. Altered cellular distribution of the iron in the heart may be a contributing factor in the develop- ment of cardiomyopathy, and may change in the absence of lymphocytes, as was observed in the liver of Rag1 single-knockout mice after dietary overloading. In the  2mRag1 double-knockout mice, dietary iron loading is not necessary because the  2m mutation leads to iron overload already under normal conditions. When fed an iron-supplemented diet,  2mRag1 double-knockout mice, like  2m -single and HFE Ϫ / Ϫ mice, 17 have a significantly lower capacity to store iron in the spleens when compared with B6 control mice on the same diet. This is partially because of the absence of a functional HFE -  2m complex , which could lead to defective storage of iron in reticuloendothelial cells. Importantly, HH patients have been reported to have a defect in iron storage in reticuloendothelial cells. 18 –20 The lower capacity to store iron may be aggravated by the lack of lymphocytes. 21,22 The lack of lymphocytes alone in Rag1 deficient mice leads to an aberrant storage of iron exclusively in parenchymal cells on dietary iron overload, in- dicating that lymphocytes may influence the iron storage capacity of reticuloendothelial cells. As a consequence of the deficient iron metabolism in the double-mutant mice, excess iron is progressively de- posited in the liver, heart, and pancreas. Thus, dietary iron overload in double-mutant mice leads to an exacer- bation of the pattern of tissue iron deposition observed when the mice are kept on a standard diet. Iron deposition in the hearts of  2mRag1 double- knockout mice, presumably leading to fibrosis, deserves special attention because heart failure is the most impor- tant life-threatening situation in untreated HH and in secondary hemochromatosis. 12–16 To our knowledge, exper- imentally induced iron-related cardiomyopathy has never been reported before in mice. Cardiac manifestations are apparent in ϳ 20% to 30% of patients presenting with clinical manifestations of HH. In younger patients they are often the presenting feature and almost always the cause of early death unless the iron is removed. In both HH and secondary hemochromatosis the iron is found predominantly within myocytes, leading to ...
Context 2
... pancreas, and heart in mice fed a standard diet and in dietary iron-loaded animals. Perl’s blue-staining of liver sections from  2m -single and  2mRag1 double-knockout mice kept on a standard diet revealed the presence of excess iron, which was predominantly in parenchymal cells (data not shown). 4,5 Moderate deposits were also observed in the pancreas and the heart of 24- to 30-week-old  2mRag1 double- knockout mice, but not in the  2m -single and Rag1 single-knockout mice or B6 wild-type mice (data not shown). As previously reported for shorter loading periods, iron deposition in the liver of B6 wild-type mice fed an iron-enriched diet up to 12 weeks was particularly prominent in Kupffer cells, and was also present in parenchymal cells (Figure 2a). Surprisingly, Rag1 single-knockout mice, that supposedly have normal Kupffer cells, develop hepatic iron overload on dietary iron loading exclusively in parenchymal cells (data not shown), like HH patients and  2m Ϫ / Ϫ mice. 5 Dietary iron-loaded  2mRag1 double-knockout mice show heavy iron depositions in the livers that corresponded to the appearance of hepato- cyte clusters (Figure 2b). A remarkable iron loading was present in the pancreas and the heart of  2mRag1 double-knockout mice (Figure 2, d and f), which was not observed in control B6 (Figure 2, c and e), and  2m single-knockout mice (data not shown). Importantly, in the pancreas this prominent iron deposition was present in acinar cells (Figure 2d), and in the heart it was present in myocytes and in the interstitial tissue (Figure 2f). Examination of hearts from  2mRag1 double-knockout mice by electron microscopy revealed frequent lysosomal structures containing granular electron-dense mate- rial in the cytoplasm of myocytes (Figure 3, a and b). Similar lysosomal iron deposition was observed in mes- enchymal perivascular cells. In the pancreas, the acinar cells contained large lysosomes of moderate electron density (Figure 3c). In these lysosomes, scattered ferritin particles were present (Figure 3d). Ferritin accumulation was also evident in the cytoplasm of acinar cells. Overall, dietary iron-loaded  2mRag1 double-knockout mice develop a more severe iron burden in multiple organs than each of the single-knockout mice, indicating an additive effect of the two mutations. To exclude the possibility that anemia could account for the abnormal iron storage defect in  2mRag1 double- knockout mice, several erythroid parameters were determined. The results demonstrated that hemoglobin, hematocrit, and mean corpuscular volume were even higher in  2m -single and in  2mRag1 double-knockout mice when compared to B6 and Rag1 Ϫ / Ϫ mice fed a standard diet (Table 2). We observed an increase of hemoglobin, hematocrit, and mean corpuscular volume values to a similar extent when B6 and Rag1 Ϫ / Ϫ mice were fed the iron-enriched diet for 12 weeks. Thus, the excess storage iron found in  2mRag1 double-knockout mice could not be attributed to defective erythropoiesis or hemoglobin synthesis. To investigate the effect of the Rag1 mutation on the absorption of iron, ferric iron, Fe(III), absorption 6 was measured before and after feeding an iron-enriched diet for 14 days (Figure 4). Ferric iron absorption after this treatment significantly decreased in all mouse strains ( P Ͻ 0.0001). However, iron absorption in  2m -single and  2mRag1 double-knockout mice was persistently higher, before and after treatment, when compared to wild-type (B6) or Rag1 single-knockout mice ( P Ͻ 0.0001, Figure 4). No significant differences were found between iron absorption in  2m -single and  2mRag1 double- knockout mice, indicating that the Rag1 mutation has no further influence on iron absorption in the gut. Iron deposition in the heart deserves special interest, because heart failure is a frequent cause of death in untreated HH and posttransfusional secondary hemochromatosis. 12–16 Remarkably, 17 out of 21  2mRag1 double-knockout mice aged between 20 and 28 weeks and kept on a standard diet developed heart fibrosis, as detected by azan staining, which was never seen in  2m and Rag1 -single-knockout mice or control mice of the same age and kept on the standard diet (Figure 5, a and b). Only after feeding an iron-enriched diet for 3 months, heart fibrosis was additionally observed in Rag1 single- knockout mice, but not in  2m single-knockout or B6 wild-type mice (data not shown). Previously we have demonstrated that reconstitution of  2m Ϫ / Ϫ mice with normal hematopoietic cells, redistrib- utes the iron from parenchymal to Kupffer cells in the liver. 5 To further investigate the influence of hematopoietic cells in the development of iron-related heart fibrosis, we reconstituted lethally irradiated 8-week-old  2mRag1 double-knockout mice with fetal liver-derived hematopoietic progenitor cells from normal mice. All reconstituted  2mRag1 double-knockout mice ( n ϭ 4) showed a normal histology up to 36 weeks of age (Figure 5 c ). The control  2mRag1 double-knockout mice reconstituted with  2mRag1 Ϫ / Ϫ -derived cells ( n ϭ 5) were sacrificed between 20 to 28 weeks when they became ill and had developed extensive fibrosis in the heart (Figure 5d). Thus, wild-type hematopoietic cell transfer prevents the development of heart fibrosis in  2mRag1 double-knockout mice. The aim of this study was to investigate the modifying influence of lymphocytes in the pathology of iron overload. Such a modifying role has been suggested by the association between low numbers of T lymphocytes in patients with HH and a more severe clinical expression of iron overload. 8 –10 In the  2m -deficient mice that develop a progressive iron overload similar to that seen in HH patients, 4 –7 we introduced the Rag1 mutation, 11 to create a total absence of mature lymphocytes. When kept on a standard diet, the double-knockout mice develop a more severe phenotype than the  2m deficient mice, involving increased iron accumulation in the liver, heart, and pancreas. The  2mRag1 double- knockout mice have visible iron depositions specifically in parenchymal cells of the liver and significantly higher iron levels in the heart than single-knockout and control mice. This indicates that the additional absence of lymphocytes, in the  2m model of iron overload, exacerbates the accumulation of iron in target organs, especially the heart. Moreover, the double-deficient mice spontane- ously develop fibrosis in the heart. The observed phenotype in the double-deficient mice is also an accentuation of the phenotype of the Rag1 single-knockout mice, which can normally regulate iron absorption and storage, and do not develop heart fibrosis under standard conditions. Rag1 single-knockout mice will develop heart fibrosis after very long periods of dietary iron loading of at least 12 weeks. Thus, dietary iron loading in combination with the lack of lymphocytes leads to cardiomyopathy. Altered cellular distribution of the iron in the heart may be a contributing factor in the develop- ment of cardiomyopathy, and may change in the absence of lymphocytes, as was observed in the liver of Rag1 single-knockout mice after dietary overloading. In the  2mRag1 double-knockout mice, dietary iron loading is not necessary because the  2m mutation leads to iron overload already under normal conditions. When fed an iron-supplemented diet,  2mRag1 double-knockout mice, like  2m -single and HFE Ϫ / Ϫ mice, 17 have a significantly lower capacity to store iron in the spleens when compared with B6 control mice on the same diet. This is partially because of the absence of a functional HFE -  2m complex , which could lead to defective storage of iron in reticuloendothelial cells. Importantly, HH patients have been reported to have a defect in iron storage in reticuloendothelial cells. 18 –20 The lower capacity to store iron may be aggravated by the lack of lymphocytes. 21,22 The lack of lymphocytes alone in Rag1 deficient mice leads to an aberrant storage of iron exclusively in parenchymal cells on dietary iron overload, in- dicating that lymphocytes may influence the iron storage capacity of reticuloendothelial cells. As a consequence of the deficient iron metabolism in the double-mutant mice, excess iron is progressively de- posited in the liver, heart, and pancreas. Thus, dietary iron overload in double-mutant mice leads to an exacer- bation of the pattern of tissue iron deposition observed when the mice are kept on a standard diet. Iron deposition in the hearts of  2mRag1 double- knockout mice, presumably leading to fibrosis, deserves special attention because heart failure is the most impor- tant life-threatening situation in untreated HH and in secondary hemochromatosis. 12–16 To our knowledge, exper- imentally induced iron-related cardiomyopathy has never been reported before in mice. Cardiac manifestations are apparent in ϳ 20% to 30% of patients presenting with clinical manifestations of HH. In younger patients they are often the presenting feature and almost always the cause of early death unless the iron is removed. In both HH and secondary hemochromatosis the iron is found predominantly within myocytes, leading to degeneration and fibrosis, with disturbances of cardiac rhythm and eventually death. 12–16,23 The typical deposition of iron in myocytes and the associated tissue damage has been difficult to mimic in animal models. In rats, after regular feeding of carbonyl iron 24 or the more efficient trimethylhexanoyl-ferrocene, 25 modest iron deposits are found in endothelial cells and perivascular macrophages. In these animal models, no stainable iron is found in myocytes and cellular damage does not occur. The mechanism by which excess iron in myocytes causes damage may involve oxidative stress and the con- secutive alteration of myocyte functions, through the iron- catalyzed Fenton chemistry. 26,27 The reason why the heart is the first organ to be affected may relate ...
Context 3
... deposition in the heart deserves special interest, because heart failure is a frequent cause of death in untreated HH and posttransfusional secondary hemo- chromatosis. [12][13][14][15][16] Remarkably, 17 out of 21 2mRag1 double-knockout mice aged between 20 and 28 weeks and kept on a standard diet developed heart fibrosis, as detected by azan staining, which was never seen in 2m- and Rag1-single-knockout mice or control mice of the same age and kept on the standard diet ( Figure 5, a and b). Only after feeding an iron-enriched diet for 3 months, heart fibrosis was additionally observed in Rag1 single- knockout mice, but not in 2m single-knockout or B6 wild-type mice (data not shown). ...
Context 4
... To further investigate the influence of hematopoi- etic cells in the development of iron-related heart fibrosis, we reconstituted lethally irradiated 8-week-old 2mRag1 double-knockout mice with fetal liver-derived hematopoi- etic progenitor cells from normal mice. All reconstituted 2mRag1 double-knockout mice (n 4) showed a nor- mal histology up to 36 weeks of age (Figure 5c). The control 2mRag1 double-knockout mice reconstituted with 2mRag1 / -derived cells (n 5) were sacrificed between 20 to 28 weeks when they became ill and had developed extensive fibrosis in the heart (Figure 5d). ...
Context 5
... reconstituted 2mRag1 double-knockout mice (n 4) showed a nor- mal histology up to 36 weeks of age (Figure 5c). The control 2mRag1 double-knockout mice reconstituted with 2mRag1 / -derived cells (n 5) were sacrificed between 20 to 28 weeks when they became ill and had developed extensive fibrosis in the heart (Figure 5d). Thus, wild-type hematopoietic cell transfer prevents the development of heart fibrosis in 2mRag1 double-knock- out mice. ...
Similar publications
To investigate the function of NF-kappa B RelA (p65), we generated mice deficient in this NF-kappa B family member by homologous recombination. Mice lacking RelA showed liver degeneration and died around embryonic day 14.5. To elucidate the role of RelA in lymphocyte development and function, we transplanted fetal liver cells of 13.5-day embryos fr...
Originally published as Nature 402,
304–309; 1999Bone remodelling and bone loss are controlled by a balance between the
tumour necrosis factor family molecule osteoprotegerin ligand (OPGL) and its
decoy receptor osteoprotegerin (OPG)1, 2, 3. In addition, OPGL
regulates lymph node organogenesis, lymphocyte development and interactions
between T cel...
The XLR gene family consists of approximately 10 X-linked genes, the expression of which is regulated in lymphocyte development. Certain members of the gene family are closely linked to the murine xid immune deficiency mutation. Sequence analysis of a cDNA clone pM1 derived from the plasmacytoma MOPC167 showed an open reading frame capable of codin...
Interleukin-7 (IL-7) is a non-hematopoietic cell-derived cytokine with a central role in the adaptive immune system. It promotes lymphocyte development in the thymus and maintains survival of naive and memory T cell homeostasis in the periphery. Moreover, it is important for the organogenesis of lymph nodes (LN) and for the maintenance of activated...
Citations
... Previous studies in mice and humans demonstrated that microbeads alone or stem cells isolated by MACS and therefore decorated with microbeads showed no deleterious effects on cardiac regeneration after cardiac injury [29], [45]. In our experiments we injected 200,000 isolated CECs corresponding to iron amounts below 60 ng and this is much less than those iron amounts that showed toxic effects on the heart before [46][47]. While in vitro studies demonstrated that microbeads, at least on non-dividing cells, persist on the cell surface for up to two weeks [48], MACsorted stem cells have been reported to lose their beads within 48 h of transplantation into myocardial infarction [29] and most likely enter the individual´s iron metabolism. ...
Background: The regenerative potential of the heart after injury is limited. Therefore, cell replacement strategies have been developed. However, the engraftment of transplanted cells in the myocardium is very inefficient. In addition, the use of heterogeneous cell populations precludes the reproducibility of the outcome.
Methods: To address both issues, in this proof of principle study, we applied magnetic microbeads for combined isolation of eGFP⁺ embryonic cardiac endothelial cells (CECs) by antigen-specific magnet-associated cell sorting (MACS) and improved engraftment of these cells in myocardial infarction by magnetic fields.
Results: MACS provided CECs of high purity decorated with magnetic microbeads. In vitro experiments revealed that the angiogenic potential of microbead-labeled CECs was preserved and the magnetic moment of the cells was strong enough for site-specific positioning by a magnetic field. After myocardial infarction in mice, intramyocardial CEC injection in the presence of a magnet resulted in a strong improvement of cell engraftment and eGFP⁺ vascular network formation in the hearts. Hemodynamic and morphometric analysis demonstrated augmented heart function and reduced infarct size only when a magnetic field was applied.
Conclusion: Thus, the combined use of magnetic microbeads for cell isolation and enhanced cell engraftment in the presence of a magnetic field is a powerful approach to improve cell transplantation strategies in the heart.
... Insoluble fibrils of β2-microglobulin (Β2-MG) are involved in dialysis-related amyloidosis [16,17]. Β2-MG is also associated with inflammation and fibrosis in the liver, heart, and kidney [18][19][20][21][22][23][24][25][26][27]. Notably, amyloid deposits have been noted in the synovium of patients undergoing HD with CTS [28][29][30]. ...
Background
Although carpal tunnel syndrome (CTS) is frequently observed in patients undergoing long-term hemodialysis (HD), exactly how CTS arises is unknown. Here, we examined levels of COL5A1 in the subsynovial connective tissue (SSCT) of patients receiving HD and studied its potential regulation by β2-microglobulin (Β2-MG) in SSCT-derived cells (SSCTCs).
Methods
We extracted SSCT samples from 67 patients with CTS (49 non-HD and 18 HD) during carpal tunnel release. The samples were subjected to quantitative polymerase chain reaction (qPCR) to determine COL5A1 expression. Further, to examine the potential regulation of COL5A1 expression by Β2-MG, SSCTCs were stimulated in the absence (control) or presence of 10 µg/ml Β2-MG.
Results
The HD group showed significantly elevated COL5A1 levels compared to the non-HD group (P=0.027). Moreover, treating SSCTCs with Β2-MG for 24 h increased the mRNA expression of COL5A1 relative to control conditions (P=0.013).
Conclusions
Elevated COL5A1 expression may form part of the mechanism underlying the development of CTS, and Β2-MG may play a role in promoting COL5A1 expression in HD patients.
... One type, known as dialysis-related amyloidosis, involves the laying down of insoluble brils of β2-microglobulin (B2M) [12,13]. According to several reports, B2M may act as an in ammatory cytokine, promoter of aging, and brosis-related factor [14][15][16][17][18][19]. Further, B2M plays a role in the formation of brosis in the kidney, heart, and liver [20][21][22][23]. Importantly, amyloid deposits are present in the synovial tissue of patients receiving dialysis with CTS [24][25][26]. ...
Purpose: Carpal tunnel syndrome (CTS) constitutes a typical complication of patients receiving long-term hemodialysis (HD). However, the mechanisms underlying the manifestation of CTS in patients receiving long-term hemodialysis remain to be studied. We investigated collagen gene expression in the subsynovial connective tissue (SSCT) of patients undergoing hemodialysis and evaluated the effect of β2-microglobulin (B2M) on collagen gene expression in cells derived from the SSCT.
Methods: To study the expression of collagen genes in the SSCT of HD and non-HD patients, SSCT specimens were obtained from 64 patients with CTS (47 non-HD and 17 HD) during carpal tunnel release (CTR). To evaluate the fibrotic condition of SSCTs, COL1A1 and COL3A1 expression were evaluated using qPCR. To evaluate the effect of B2M on collage genes, SSCT cells were stimulated in α-MEM with 0.5% fetal bovine (vehicle) or 1 or 10 µg/ml B2M.
Results: Expressions of both COL1A1 and COL3A1 in the HD group were significantly higher than those in the non-HD group (COL1A1, P=0.021; COL3A1, P=.0.031). Incubating SSCT cells together with B2M for 6 h and 24 h led to increased mRNA expression of COL3A1 compared to that observed in vehicle control cells (p<0.001
Conclusions: Our findings suggest that increased expression of COL1A1 and COL3A1 may be one mechanism by which HD patients develop CTS. Further, B2M may be responsible for elevating COL3A1 expression in HD patients with CTS.
... bw, body weight; RBC, red blood cell; ROS, reactive oxygen species Serum iron, Tf saturation, total iron-binding capacity, and unbound iron-binding capacity were determined using commercially available assay kits (Nanjing Jincheng Bioengineering Institute). Tissue iron levels were determined as previously described (80). Tissue samples were dried at 106 C overnight to constant mass and weighed again. ...
Iron accumulates with age in mammals, and its possible implications in altering metabolic responses are not fully understood. Here we report that both high-iron diet and advanced age in mice consistently altered gene expression of many pathways, including those governing the oxidative stress response and the circadian clock. We used a metabolomic approach to reveal similarities between metabolic profiles and the daily oscillation of clock genes in old and iron-overloaded mouse livers. In addition, we show that phlebotomy decreased iron accumulation in old mice, partially restoring the metabolic patterns and amplitudes of the oscillatory expression of clock genes Per1 and Per2. We further identified that the transcriptional regulation of iron occurred through a reduction in AMP-modulated methylation of histone H3K9 in the Per1 and H3K4 in the Per2 promoters, respectively. Taken together, our results indicate that iron accumulation with age can affect metabolic patterns and the circadian clock.
... β2-Microglobulin (β2M) is the light chain of the class I major histocompatibility complex (MHC I) protein that plays an important role in innate and adaptive immunity, and is involved in the pathogenesis of respiratory diseases [1,2]. Some studies have suggested that β2M may function as an inflammatory cytokine, aging-promoting factor, and fibrosis-related factor [3][4][5][6][7][8]. In addition, β2M is involved in the development of fibrosis in kidney, heart, and liver [9][10][11][12]. ...
... Due to β2M structural characteristics, this amyloid protein can be distributed by blood to all parts of the body, deposit in various tissues and organs, and cause varying degrees of destruction [22]. β2M has been used as a biomarker of fibrosis progression in several organs, including kidney, heart, and liver [6,7,9,12]. However, its role in the lungs has been insufficiently studied. ...
Expression of β2-microglobulin (β2M) is involved in fibrosis progression in kidney, liver, and heart. In this case-controlled retrospective study, we investigated the role of β2M in the development of pulmonary fibrosis in patients with chronic obstructive pulmonary disease (COPD). Analysis of 450 COPD patients revealed that patients with decreased pulmonary diffusing capacity (DLCO) had increased β2M serum levels. Compared to patients with lower β2M serum levels, patients with increased β2M levels exhibited increased alveolar wall/septal thickening and lung tissue β2M expression. In addition, patients with increased β2M levels had increased lung expression of TGF-β1, Smad4, and a-SMA. Animal experiments showed that increased β2M expression resulted in epithelial-mesenchymal transition (EMT), alveolar wall/septal thickening, and pulmonary fibrosis in a rat COPD model. Together, these results indicate that β2M serum levels may serve as a new indicator for assessment of pulmonary diffusion function and pulmonary fibrosis severity in clinical practice and may provide a potential target for treatment of pulmonary fibrosis in the future.
... When those defects were described, they generated some surprise and confusion amongst researchers and clinicians because of the previously existing evidence of iron induced expansions of T lymphocyte populations, namely, relative expansions of CD8+ T lymphocytes, both in experimental models of Fe-citrate injection [22,23] and in clinical models of transfusional iron overload [24,25]. The reciprocal effect, i.e., that primary immune defects could, in turn, contribute to iron overload, was next confirmed with a number of experimental studies examining and confirming the presence of iron overload in mice Pharmaceuticals 2019, 12, 122 3 of 12 deficient in selected [26][27][28] or total lymphocytes [29] and a more severe phenotype in mice lacking both HFE and β2-microglobulin [30]. Surprisingly, mice lacking only classical MHC class I molecules also developed iron overload [31]. ...
The HFE gene (OMIM 235200), most commonly associated with the genetic iron overload disorder Hemochromatosis, was identified by Feder et al. in 1996, as a major histocompatibilty complex (MHC) class I like gene, first designated human leukocyte antigen-H (HLA-H). This discovery was thus accomplished 20 years after the realization of the first link between the then “idiopathic” hemochromatosis and the human leukocyte antigens (HLA). The availability of a good genetic marker in subjects homozygous for the C282Y variant in HFE (hereditary Fe), the reliability in serum markers such as transferrin saturation and serum ferritin, plus the establishment of noninvasive methods for the estimation of hepatic iron overload, all transformed hemochromatosis into a unique age related disease where prevention became the major goal. We were challenged by the finding of iron overload in a 9-year-old boy homozygous for the C282Y HFE variant, with two brothers aged 11 and 5 also homozygous for the mutation. We report a 20 year follow-up during which the three boys were seen yearly with serial determinations of iron parameters and lymphocyte counts. This paper is divided in three sections: Learning, applying, and questioning. The result is the illustration of hemochromatosis as an age related disease in the transition from childhood to adult life and the confirmation of the inextricable link between iron overload and the cells of the immune system.
... Animal models support a link between cardiac iron and cardiac fibrosis. Cardiac fibrosis was prominent in double knock-out mice for beta 2 microglobulin (B2m, deficiency of which causes increased gut uptake of iron through impairment of the HFE-B2m complex) and recombinase activator gene 1 (Rag1, deficiency of which causes absence of B and T lymphocytes) which was not seen in B2m and Rag1 single-knockout mice or control mice of the same age, implying that lymphocytes play a role in cardiac fibrosis which is additive to cardiac iron loading alone [37]. Other iron loading animal models also show cardiac fibrosis, although this was not prominent [38,39]. ...
Background
Heart failure related to cardiac siderosis remains a major cause of death in transfusion dependent anaemias. Replacement fibrosis has been reported as causative of heart failure in siderotic cardiomyopathy in historical reports, but these findings do not accord with the reversible nature of siderotic heart failure achievable with intensive iron chelation.
Methods
Ten whole human hearts (9 beta-thalassemia major, 1 sideroblastic anaemia) were examined for iron loading and fibrosis (replacement and interstitial). Five had died from heart failure, 4 had cardiac transplantation for heart failure, and 1 had no heart failure (death from a stroke). Heart samples iron content was measured using atomic emission spectroscopy. Interstitial fibrosis was quantified by computer using picrosirius red (PSR) staining and expressed as collagen volume fraction (CVF) with normal value for left ventricle <3%.
Results
The 9 hearts affected by heart failure had severe iron loading with very low T2* of 5.0???2.0?ms (iron concentration 8.5???7.0?mg/g dw) and diffuse granular myocardial iron deposition. In none of the 10 hearts was significant macroscopic replacement fibrosis present. In only 2 hearts was interstitial fibrosis present, but with low CVF: in one patient with no cardiac siderosis (death by stroke, CVF 5.9%) and in a heart failure patient (CVF 2%). In the remaining 8 patients, no interstitial fibrosis was seen despite all having severe cardiac siderosis and heart failure (CVF 1.86% ?0.87%).
Conclusion
Replacement cardiac fibrosis was not seen in the 9 post-mortem hearts from patients with severe cardiac siderosis and heart failure leading to death or transplantation, which contrasts markedly to historical reports. Minor interstitial fibrosis was also unusual and very limited in extent. These findings accord with the potential for reversibility of heart failure seen in iron overload cardiomyopathy.
Trial registration
ClinicalTrials.gov Identifier: NCT00520559
... Beyond HH: Perspectives on the Immunological Role of HFE Although HFE cannot present antigens, it actively participates in the MHC I pathway and CD8 þ T cell activation, revealing an immunological role as a negative regulator of MHC I antigen presentation. HFE association with T cells was also previously highlighted with animal studies that demonstrated that iron overload is more prominent in RAG1 mice deficient in lymphocytes and in HFE-deficient mice on a RAG1 background [125,126]. Overall, HFE reveals the close relationship between iron metabolism and immunity, and appears to act as a mediator between both processes. HFE C282Y has been associated with the UPR, a cellular stress response affecting the MHC I pathway, which may provide new clues that link UPR signaling pathways and HH pathophysiology [127]. ...
Introduction: Since its discovery, the hemochromatosis protein HFE has been primarily defined by its role in iron metabolism and homeostasis, and its involvement in the genetic disease termed hereditary hemochromatosis (HH). While HH patients are typically afflicted by dysregulated iron levels, many are also affected by several immune defects and increased incidence of autoimmune diseases that have thereby implicated HFE in the immune response. Growing evidence has supported an immunological role for HFE with recent studies describing HFE specifically as it relates to MHC I antigen presentation.
Methods/Results: Here, we present a comprehensive overview of the relationship between iron metabolism, HFE, and the immune system to better understand the origin and cause of immune defects in HH patients. We further describe the role of HFE in MHC I antigen presentation and its potential to impair autoimmune responses in homeostatic conditions, a mechanism which may be exploited by tumors to evade immune surveillance.
Conclusion: Overall, this increased understanding of the role of HFE in the immune response sets the stage for better treatment and management of hereditary hemochromatosis and other iron-related diseases, as well as of the immune defects related to this condition.
... In a haemochromatosis mouse model (Santos et al, 2000) fed a 2.5% (w/w) carbonyl iron diet for 12 weeks, a small amount of iron deposits were noted within lysosomes in the cardiomyocytes using an electron microscope. On examination of the histology Perls stain images, the majority of iron appears to be in the interstitium and there is no clear iron deposition within the cardiomyocyte. ...
Iron overload is an inevitable consequence of repeated blood transfusions required to sustain life in a wide array of haematological conditions such as thalassaemia, aplastic anaemia, and myelodysplastic syndromes (MDS). Without iron chelation therapy, death from cardiotoxic effects of iron overload usually ensues in the second decade. Iron chelation therapy with subcutaneous Desferrioxamine (DFO) infusions at least 5 nights per week has been shown unequivocally to prolong life expectancy in thalassaemia major. However, although this molecule is remarkably free of toxic side effects at treatment doses, patient compliance is often poor, and iron overload still leads to death today. One of the scopes of this Ph.D. was to develop a cellular model that would allow the testing of novel chelating agents used alone or in combination with established chelators. Our in vitro model of iron overload was able to elucidate several principles regarding the interactions of chelators within cells. It allowed for the first time a detailed interrogation of synergy as opposed to additivity of action of licensed chelators when used in combination, which has now been published. This model is also relevant to the development of new chelators and was used to demonstrate the iron binding properties of Eltrombopag (ELT), a drug used to manage ITP, at clinically achievable concentrations. ELT is a powerful intracellular iron chelator that decreases storage iron and enhances iron removal when in combination with commercially available iron chelators. In clinical use, donation of chelated iron by ELT to these chelators offers established routes for elimination of chelated iron. Furthermore, we extensively investigated the iron mobilising properties of the naturally occurring flavonoid quercetin and its principle metabolites. For the first time we showed that quercetin and its metabolites can act as a shuttle when combined with licensed chelators and provided a unique structure-function analysis of flavonoids with regards to iron and ferritn mobilisation and antioxidant capacity as a function of Fe(II) binding. A further goal of this thesis was to establish an iron-overloaded humanised thalassaemia mouse model that could be used to examine whether the same principles which determine iron release from cell cultures also influence the oral efficiency of iron chelators, in vivo. We utilised iron dextran to achieve cardiac iron loading confirmed by histology and MRI investigations. Iron overloaded mice were treated with a combination of the flavonoid quercetin and the iron chelator Deferasirox (DFX), and we established the value of this in combination in terms of cardiac iron mobilisation. Our novel humanised β thalassaemia ion-overloaded mouse model demonstrating cardiac iron loading is a first-in-kind development, and the novel application of MRI will provide a useful tool for studying iron chelators, the pathophysiology and disease progression, blood transfusion regimens and cellular/gene therapy in iron overload in the future. Our findings in vivo support the contention that our cellular model is a useful screening tool for new compounds, both for toxicity and efficacy.
... However, there is evidence that suggests a potential immunological role for HFE as well. For example, the iron overload phenotype is even more pronounced in mice that are deficient for both β2m and RAG1, compared to β2m −/− animals (Santos et al. 2000). HFE-deficient animals on a RAG1 background also have increased iron overload, compared to HFE −/− animals (Miranda et al. 2004). ...
Even though major histocompatibility complex (MHC) class Ia and many Ib molecules have similarities in structure, MHC class Ib molecules tend to have more specialized functions, which include the presentation of non-peptidic antigens to non-classical T cells. Likewise, non-classical T cells also have unique characteristics, including an innate-like phenotype in naïve animals and rapid effector functions. In this review, we discuss the role of MAIT and NKT cells during infection but also the contribution of less studied MHC class Ib-restricted T cells such as Qa-1-, Qa-2-, and M3-restricted T cells. We focus on describing the types of antigens presented to non-classical T cells, their response and cytokine profile following infection, as well as the overall impact of these T cells to the immune system.
