Expression of human equilibrative nucleoside transporter 1 (hENT1) in pancreatic ductal adenocarc... more Expression of human equilibrative nucleoside transporter 1 (hENT1) in pancreatic ductal adenocarcinoma (PDAC) has been postulated to be a marker of sensitivity to gemcitabine. However, heterogeneity in the studies attempting to quantify hENT1 expression in patients with PDAC treated with gemcitabine has yielded inconclusive results that impede the adoption of hENT1 expression as a predictive biomarker. Tissue microarrays consisting of PDAC specimens from 227 patients acquired between 1987 and 2013 annotated with treatment and outcome information were subjected to staining with two antibodies for hENT1 (10D7G2 and SP120) on a single automated platform and scored by two independent pathologists blinded to treatment and outcome. The resultant scores were subjected to individual predictive disease-specific survival analysis and to unsupervised hierarchical clustering to generate a multi-marker classification. Tumour cell staining prevalence using either SP120 or 10D7G2 was predictive of gemcitabine sensitivity (p 5 0.02; p 5 0.01). When combined, three groups emerged, classified as SP120 Low _10D7G2 Low , SP120 Low _10D7G2 High , and SP120 High _10D7G2 High , in which adjuvant gemcitabine conferred median survival differences of 0.2, 0.8, and 1.5 (p 5 0.76, p 5 0.06, p 5 0.01) years, respectively. These results were largely replicated in multivariable analysis with the P value for the SP120 Low _10D7G2 High cluster achieving statistical significance (p 5 0.03). These data suggest that either antibody for hENT1 can be used to predict gemcitabine sensitivity in resected PDAC. However, using both antibodies adds valuable information that enables the stratification of patients who can expect to have a good, intermediate, and poor response to adjuvant gemcitabine.
While short-term graft survival rates among pediatric kidney transplant recipients have improved ... more While short-term graft survival rates among pediatric kidney transplant recipients have improved over the years, long-term survival is still limited by chronic allograft injury. 1-3 Pediatric kidney transplant recipients have higher immunological responsiveness compared to adult recipients, which may place them at higher risk of developing TCMR. 4-7 Functional monitoring of serum Cr to identify acute
Oxidized LDL (OxLDL) is thought to play a role in the pathogenesis of early as well as advanced s... more Oxidized LDL (OxLDL) is thought to play a role in the pathogenesis of early as well as advanced stages of atherosclerosis. One possible mechanism involves local upregulation of pro-inflammatory cytokines such as vascular endothelial growth factor (VEGF). This study was done to define the mechanism by which OxLDL increases secretion of VEGF in macrophages. The murine leukemia-derived RAW 264.7 macrophage cell line as well as mouse peritoneal macrophages and human monocyte-derived macrophages were used in these studies. Cells were exposed to native lowdensity lipoprotein (LDL), acetylated LDL, and LDL that had been modified by oxidation with copper or ferrous ions or by exposure to auto-oxidation products of arachidonic acid for 16 h, and VEGF was then assayed in medium. Pharmacological inhibitors of phosphatidylinositol 3-kinase (PI3K) or PKC blocked VEGF secretion by OxLDL. Inhibitors of other protein kinase C (PKC) subtypes had no effect, and neither did inhibitors of mitogen activated protein kinase kinase (MAPKK). We found that LDL with oxidative modification of either its lipid or protein component can induce VEGF expression. Higher degrees of oxidation of LDL conferred higher potency to induce VEGF. Macrophages from mice lacking both scavenger receptors A (SR-A) and CD36 were fully responsive to OxLDL with regard to VEGF secretion. These macrophages show an 85% reduction in OxLDL uptake compared to macrophages from wild-type mice. Macrophages from mice lacking LOX-1 were also fully responsive to oxLDL with regard to VEGF secretion. We conclude that VEGF upregulation is mediated through PI3K and PKC, and does not involve the above three scavenger receptors or require uptake of oxidized LDL.
role in atherogenesis, in part because of its effects on macrophage recruitment and retention (7,... more role in atherogenesis, in part because of its effects on macrophage recruitment and retention (7, 8). Initial oxidation of LDL and formation of what is often referred to as "minimally modifi ed" LDL stimulate adjacent endothelial and smooth muscle cells to release monocyte chemotactic protein-1, which facilitates the recruitment of monocytes into the arterial wall. OxLDL itself is chemotactic for monocytes by virtue of its lysophosphatidylcholine content. At high concentrations, oxLDL can be toxic to cultured macrophages and other cells, but at lower concentrations, it has been clearly shown to promote macrophage proliferation and inhibit apoptosis (9-15). Our group has recently reported that oxLDL inhibits macrophage apoptosis by activating eukaryotic elongation factor-2 (eEF2) kinase (also known as Ca 2+ /calmodulin-dependent kinase III) (16). eEF2 kinase activation and inhibition of macrophage apoptosis is mediated by an oscillatory increase in intracellular calcium ([Ca 2+ ] i). However, the signal transduction pathways involved in oxLDL-mediated [Ca 2+ ] i oscillations have not been elucidated. Ca 2+ is a ubiquitous intracellular signal responsible for controlling numerous cellular processes. These processes range from muscle contraction to synaptic transmission and from cellular proliferation to apoptosis (17). Ca 2+ can relay specifi city in signaling through its high degree of spatial and temporal diversity (18). Ca 2+ released into the cytoplasm can function as a second messenger that can Abstract We recently reported that oxidized LDL (oxLDL) induces an oscillatory increase in intracellular calcium ([Ca 2+ ] i) levels in macrophages. Furthermore, we have shown that these [Ca 2+ ] i oscillations mediate oxLDL's ability to inhibit macrophage apoptosis in response to growth factor deprivation. However, the signal transduction pathways by which oxLDL induces [Ca 2+ ] i oscillations have not been elucidated. In this study, we show that these oscillations are mediated in part by intracellular mechanisms, as depleting extracellular Ca 2+ did not completely abolish the effect. Inhibiting sarco-endoplasmic reticulum ATPase (SERCA) completely blocked [Ca 2+ ] i oscillations, suggesting a role for Ca 2+ reuptake by the ER. The addition of oxLDL resulted in an almost immediate activation of sphingosine kinase (SK), which can increase sphingosine-1-phosphate (S1P) levels by phosphorylating sphingosine. Moreover, S1P was shown to be as effective as oxLDL in blocking macrophage apoptosis and producing [Ca 2+ ] i oscillations. This suggests that the mechanism in which oxLDL generates [Ca 2+ ] i oscillations may be 1) activation of SK, 2) SK-mediated increase in S1P levels, 3) S1P-mediated Ca 2+ release from intracellular stores, and 4) SERCA-mediated Ca 2+ reuptake back into the ER.
Macrophages are prominent components of human atherosclerotic lesions and they are believed to ac... more Macrophages are prominent components of human atherosclerotic lesions and they are believed to accelerate the progression and/or complications of both early and advanced atherosclerotic lesions. We and others have shown that oxidized low-density lipoprotein (oxLDL) induces growth and inhibits apoptosis in murine bone marrow-derived macrophages. In this study, we sought to characterize the oxidative modification of LDL that is responsible for this prosurvival effect. We found that both the modified lipid and the modified protein components of oxLDL can increase the viability of macrophages. The key modification appeared to involve derivatization of amino groups in apoB or in phosphatidylethanolamine by lipid peroxidation products. These reactive oxidation products were primarily unfragmented hydroperoxide- or endoperoxide-containing oxidation products of linoleic acid or arachidonic acid. LC-MS/MS studies showed that some of the arachidonic acid-derived lysine adducts were isolevuglandins that contain lactam and hydroxylactam rings. MS/MS analysis of linoleic acid autoxidation adducts was consistent with 5- or 6-membered nitrogen-containing heterocycles derived from unfragmented oxidation products. The amine modification by oxidation products generated a fluorescence pattern with an excitation maximum at 350nm and emission maximum at 430nm. This is very similar to the fluorescence spectrum of copper-oxidized LDL.
The bioactive sphingolipid ceramide 1-phosphate (C1P) regulates cell division in a variety of cel... more The bioactive sphingolipid ceramide 1-phosphate (C1P) regulates cell division in a variety of cell types including macrophages. However, the mechanisms involved in this action are not completely understood. In the present work we show that C1P stimulates the release of vascular endothelial growth factor (VEGF) in RAW264.7 macrophages, and that this growth factor is essential for stimulation of cell proliferation by C1P. The stimulation of VEGF release was dependent upon activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB-1 also known as Akt-1), and mitogen-activated protein kinase-kinase (MEK)/extracellularly regulated kinase-2 (ERK-2) pathways, as inhibition of these kinases with selective pharmacological inhibitors or with specific gene silencing siRNA, abrogated VEGF release. A key observation was that sequestration of VEGF with a neutralizing antibody, or treatment with VEGF siRNA abolished C1P-stimulated macrophage growth. Also, inhibition of the pathways involved in C1P-stimulated VEGF release inhibited the stimulation of macrophage growth by C1P. Moreover, blockade of VEGF receptor-2 (VEGFR-2), which is the primary receptor for VEGF, with the pharmacological inhibitor DMH4, or with specific VEGFR-2 siRNA, substantially inhibited C1P-stimulated cell growth. It can be concluded that stimulation of VEGF release is a key factor in the promotion of macrophage proliferation by C1P.
International Journal of Nephrology and Renovascular Disease, Jun 1, 2022
Lipoprotein glomerulopathy (LPG) is caused by a mutation in the apolipoprotein E gene (APOE) gene... more Lipoprotein glomerulopathy (LPG) is caused by a mutation in the apolipoprotein E gene (APOE) gene and is characterized by lipoprotein thrombi in glomerular capillaries. Here, we describe a case of LPG, the first to be reported from Canada and the first case of LPG in North America to be associated with the APOE Tokyo/Maebashi mutation (p.Leu162_Lys164del, traditional nomenclature 142_144del). A 49-year-old man of Chinese descent with a previous diagnosis of dyslipidemia and a new diagnosis of hypertension was found to have proteinuria on routine urinalysis. Renal biopsy showed markedly dilated glomerular capillaries filled with pale staining mesh-like material that stained positive for Oil-RedO , consistent with lipoprotein thrombi. APOE gene sequencing confirmed the diagnosis of LPG. The patient was treated with fenofibrate and perindopril. His lipid profile normalized and proteinuria dropped to minimal levels. Repeat renal biopsy 2 years after the first showed resolution of lipoprotein thrombi but with rare residual granular densities by electron microscopy consistent with lipoprotein in the subendothelial space, supporting the hypothesis that this subendothelial material contains precursors to lipoprotein thrombi.
We tested whether loss of eukaryotic elongation factor 2 kinase (eEF2K) activity in macrophages s... more We tested whether loss of eukaryotic elongation factor 2 kinase (eEF2K) activity in macrophages suppresses development of atherosclerosis by transplanting bone marrow from mice with mutant eEF2K into ldlr Ă/Ă mice. Sixteen weeks after high-fat diet feeding, mutant eEF2K hematopoietic chimeras had a dramatically reduced level of atherosclerotic plaque formation. M1-skewed macrophages from eEF2K knock-in mice have less tumour necrosis factor-a release and a lesser ability to induce expression of endothelial cell markers, providing a potential explanation for the role of eEF2K. Because eEF2K activity in cells of the hematopoietic compartment contributes to atherosclerosis development, drugs inhibiting eEF2K might have a beneficial effect in treatment of atherosclerosis.
Clinical Journal of The American Society of Nephrology, Nov 4, 2019
Background and objectives Fibrillary GN has been defined as an immune complex-mediated GN with am... more Background and objectives Fibrillary GN has been defined as an immune complex-mediated GN with amyloidlike fibrils larger than amyloid which are IgG positive and Congo red negative. With discovery of DNAJB9 as a highly sensitive and specific marker for fibrillary GN, the specificity of the morphologic criteria for establishing the diagnosis of fibrillary GN has come into question. Design, setting, participants, & measurements We sought to (1) determine anatomic characteristics that best define fibrillary GN and (2) identify clinical and pathologic features that predict outcomes. Results We retrospectively reviewed kidney biopsies from patients diagnosed with fibrillary GN or suspected fibrillary GN between 1997 and 2017 (n=266, 65% female, median age 61). Approximately 11% of kidney biopsies had one or more unusual feature including monotypic deposits, Congo red positivity, or unusual fibril diameter. Fibrillary GN as a possible monoclonal gammopathy of renal significance represented ,1% of cases. Immunostaining for DNAJB9 confirmed fibrillary GN in 100% of cases diagnosed as fibrillary GN and 79% of atypical cases diagnosed as possible fibrillary GN. At a median time of 24 months (interquartile range, 8-46 months) after biopsy (n=100), 53% of patients reached the combined primary outcome of ESKD or death, 18% had CKD, and 18% had partial remission. On multivariable analysis, male sex (adjusted hazard ratio [aHR], 3.82; 95% confidence interval [95% CI], 1.97 to 7.37) and eGFR were the most significant predictors of primary outcome (aHR of 8.02 if eGFR ,30 ml/min per 1.73 m 2 [95% CI, 1.85 to 34.75]; aHR of 6.44 if eGFR 30 to ,45 ml/min per 1.73 m 2 [95% CI, 1.38 to 29.99]). Immunosuppressive therapy with rituximab was significantly associated with stabilization of disease progression. Conclusions Detection of DNAJB9 is a useful diagnostic tool for diagnosing atypical forms of fibrillary GN. The outcomes for fibrillary GN are poor and progression to ESKD is influenced predominantly by the degree of kidney insufficiency at the time of diagnosis and male sex. Rituximab may help preserve kidney function for select patients with fibrillary GN.
Oxidized LDL (oxLDL) promotes lipid accumulation as well as growth and survival signaling in macr... more Oxidized LDL (oxLDL) promotes lipid accumulation as well as growth and survival signaling in macrophages. OxLDL uptake is mainly due to scavenger receptors SR-AI/II and CD36. However, other scavenger receptors such as lectin-like oxLDL receptor-1 (LOX-1) may also play a role. We used mice with targeted inactivation of the LOX-1 gene to defi ne the role of this receptor in the uptake of oxLDL and in activation of survival pathways. There was no difference in uptake or degradation of 125 I-oxLDL in unstimulated macrophages from wild-type and LOX-1 knockout mice and no difference in the rate of clearance of oxLDL from plasma in vivo. However, when expression of LOX-1 was induced with lysophosphatidylcholine, oxLDL uptake and degradation increased 2-fold in wild-type macrophages but did not change in LOX-1 knockout macrophages. Macrophages lacking LOX-1 showed the same stimulation of PKB phosphorylation and enhancement of survival by oxLDL as wild-type cells. These data show that LOX-1 does not alter the uptake of oxLDL in unstimulated macrophages and is not essential for the pro-survival effect of oxLDL in these cells. However, LOX-1 expression is highly inducible by lysophosphatidylcholine and pro-infl ammatory cytokines, and if that occurred in macrophages within atheromas, LOX-1 could substantially increase oxLDL uptake by lesion macrophages.
BACKGROUND Early dysfunction of renal allografts may be associated with vascular injury, which ra... more BACKGROUND Early dysfunction of renal allografts may be associated with vascular injury, which raises the specter of active rejection processes that require medical intervention. In our practice, we have encountered patients who present with delayed graft function and demonstrate a unique pattern of endothelial cell injury that raises concern for rejection in their biopsy. Therefore, we sought to systematically determine the biopsy characteristics and outcome of these patients. METHODS During a 17-year period at the University of Washington in Seattle, United States, we identified 24 cases of a distinct arterial vasculopathy presenting in the first year posttransplantation. This early transplant arteriopathy (ETA) is characterized by endothelial cell swelling and intimal edema but without the intimal arteritis that defines vascular rejection. RESULTS Approximately 1% of transplant biopsies during the study period showed ETA, almost all of which were in deceased donor organs (96%), and most presented with delayed graft function (54%) or increased serum creatinine (38%) soon after transplantation (median 13 days; range, 5-139). In this study, 77% of patients were managed expectantly, with only 2 patients (7.6%) subsequently developing acute vascular rejection. Except for 1 patient who died, all patients had functioning allografts at 1 year follow-up. CONCLUSION Recognizing ETA and distinguishing it from vascular rejection is important to prevent over-treatment because most patients appear to recover allograft function rapidly with expectant management.
Familial hypobetalipoproteinemia (FHBL) is a rare genetic disorder of lipid metabolism that is as... more Familial hypobetalipoproteinemia (FHBL) is a rare genetic disorder of lipid metabolism that is associated with abnormally low serum levels of low-density lipoprotein (LDL) cholesterol and apolipoprotein B. It is an autosomal co-dominant disorder, and depending on zygosity, the clinical manifestations may vary from none to neurological, endocrine, hematological or liver dysfunction. Nonalcoholic fatty liver disease is common in persons with FHBL, however progression to nonalcoholic steatohepatitis is unusual. We describe here a patient with a novel APOB mutation, V703I, which appears to contribute to the severity of the FHBL phenotype. He had liver enzyme abnormalities, increased echogenicity of the liver consistent with steatosis, very low LDL cholesterol at 0.24 mmol/l (normal 1.8-3.5 mmol/l) and an extremely low apolipoprotein B level of 0.16 g/l (normal 0.6-1.2 g/l). APOB gene sequencing revealed him to be a compound heterozygote with two mutations (R463W and V703I). APOB R463W has previously been reported to cause FHBL. Genetic sequencing of his first-degree relatives identified the APOB V703I mutation in his normolipidemic brother and father and the APOB R463W mutation in his mother and sister, both of whom have very low LDL cholesterol levels. These results suggest that the APOB V703I mutation alone does not cause the FHBL phenotype. However, it is possible that it has a contributory role to a more aggressive phenotype in the presence of APOB R463W.
Expression of human equilibrative nucleoside transporter 1 (hENT1) in pancreatic ductal adenocarc... more Expression of human equilibrative nucleoside transporter 1 (hENT1) in pancreatic ductal adenocarcinoma (PDAC) has been postulated to be a marker of sensitivity to gemcitabine. However, heterogeneity in the studies attempting to quantify hENT1 expression in patients with PDAC treated with gemcitabine has yielded inconclusive results that impede the adoption of hENT1 expression as a predictive biomarker. Tissue microarrays consisting of PDAC specimens from 227 patients acquired between 1987 and 2013 annotated with treatment and outcome information were subjected to staining with two antibodies for hENT1 (10D7G2 and SP120) on a single automated platform and scored by two independent pathologists blinded to treatment and outcome. The resultant scores were subjected to individual predictive disease-specific survival analysis and to unsupervised hierarchical clustering to generate a multi-marker classification. Tumour cell staining prevalence using either SP120 or 10D7G2 was predictive of gemcitabine sensitivity (p 5 0.02; p 5 0.01). When combined, three groups emerged, classified as SP120 Low _10D7G2 Low , SP120 Low _10D7G2 High , and SP120 High _10D7G2 High , in which adjuvant gemcitabine conferred median survival differences of 0.2, 0.8, and 1.5 (p 5 0.76, p 5 0.06, p 5 0.01) years, respectively. These results were largely replicated in multivariable analysis with the P value for the SP120 Low _10D7G2 High cluster achieving statistical significance (p 5 0.03). These data suggest that either antibody for hENT1 can be used to predict gemcitabine sensitivity in resected PDAC. However, using both antibodies adds valuable information that enables the stratification of patients who can expect to have a good, intermediate, and poor response to adjuvant gemcitabine.
While short-term graft survival rates among pediatric kidney transplant recipients have improved ... more While short-term graft survival rates among pediatric kidney transplant recipients have improved over the years, long-term survival is still limited by chronic allograft injury. 1-3 Pediatric kidney transplant recipients have higher immunological responsiveness compared to adult recipients, which may place them at higher risk of developing TCMR. 4-7 Functional monitoring of serum Cr to identify acute
Oxidized LDL (OxLDL) is thought to play a role in the pathogenesis of early as well as advanced s... more Oxidized LDL (OxLDL) is thought to play a role in the pathogenesis of early as well as advanced stages of atherosclerosis. One possible mechanism involves local upregulation of pro-inflammatory cytokines such as vascular endothelial growth factor (VEGF). This study was done to define the mechanism by which OxLDL increases secretion of VEGF in macrophages. The murine leukemia-derived RAW 264.7 macrophage cell line as well as mouse peritoneal macrophages and human monocyte-derived macrophages were used in these studies. Cells were exposed to native lowdensity lipoprotein (LDL), acetylated LDL, and LDL that had been modified by oxidation with copper or ferrous ions or by exposure to auto-oxidation products of arachidonic acid for 16 h, and VEGF was then assayed in medium. Pharmacological inhibitors of phosphatidylinositol 3-kinase (PI3K) or PKC blocked VEGF secretion by OxLDL. Inhibitors of other protein kinase C (PKC) subtypes had no effect, and neither did inhibitors of mitogen activated protein kinase kinase (MAPKK). We found that LDL with oxidative modification of either its lipid or protein component can induce VEGF expression. Higher degrees of oxidation of LDL conferred higher potency to induce VEGF. Macrophages from mice lacking both scavenger receptors A (SR-A) and CD36 were fully responsive to OxLDL with regard to VEGF secretion. These macrophages show an 85% reduction in OxLDL uptake compared to macrophages from wild-type mice. Macrophages from mice lacking LOX-1 were also fully responsive to oxLDL with regard to VEGF secretion. We conclude that VEGF upregulation is mediated through PI3K and PKC, and does not involve the above three scavenger receptors or require uptake of oxidized LDL.
role in atherogenesis, in part because of its effects on macrophage recruitment and retention (7,... more role in atherogenesis, in part because of its effects on macrophage recruitment and retention (7, 8). Initial oxidation of LDL and formation of what is often referred to as "minimally modifi ed" LDL stimulate adjacent endothelial and smooth muscle cells to release monocyte chemotactic protein-1, which facilitates the recruitment of monocytes into the arterial wall. OxLDL itself is chemotactic for monocytes by virtue of its lysophosphatidylcholine content. At high concentrations, oxLDL can be toxic to cultured macrophages and other cells, but at lower concentrations, it has been clearly shown to promote macrophage proliferation and inhibit apoptosis (9-15). Our group has recently reported that oxLDL inhibits macrophage apoptosis by activating eukaryotic elongation factor-2 (eEF2) kinase (also known as Ca 2+ /calmodulin-dependent kinase III) (16). eEF2 kinase activation and inhibition of macrophage apoptosis is mediated by an oscillatory increase in intracellular calcium ([Ca 2+ ] i). However, the signal transduction pathways involved in oxLDL-mediated [Ca 2+ ] i oscillations have not been elucidated. Ca 2+ is a ubiquitous intracellular signal responsible for controlling numerous cellular processes. These processes range from muscle contraction to synaptic transmission and from cellular proliferation to apoptosis (17). Ca 2+ can relay specifi city in signaling through its high degree of spatial and temporal diversity (18). Ca 2+ released into the cytoplasm can function as a second messenger that can Abstract We recently reported that oxidized LDL (oxLDL) induces an oscillatory increase in intracellular calcium ([Ca 2+ ] i) levels in macrophages. Furthermore, we have shown that these [Ca 2+ ] i oscillations mediate oxLDL's ability to inhibit macrophage apoptosis in response to growth factor deprivation. However, the signal transduction pathways by which oxLDL induces [Ca 2+ ] i oscillations have not been elucidated. In this study, we show that these oscillations are mediated in part by intracellular mechanisms, as depleting extracellular Ca 2+ did not completely abolish the effect. Inhibiting sarco-endoplasmic reticulum ATPase (SERCA) completely blocked [Ca 2+ ] i oscillations, suggesting a role for Ca 2+ reuptake by the ER. The addition of oxLDL resulted in an almost immediate activation of sphingosine kinase (SK), which can increase sphingosine-1-phosphate (S1P) levels by phosphorylating sphingosine. Moreover, S1P was shown to be as effective as oxLDL in blocking macrophage apoptosis and producing [Ca 2+ ] i oscillations. This suggests that the mechanism in which oxLDL generates [Ca 2+ ] i oscillations may be 1) activation of SK, 2) SK-mediated increase in S1P levels, 3) S1P-mediated Ca 2+ release from intracellular stores, and 4) SERCA-mediated Ca 2+ reuptake back into the ER.
Macrophages are prominent components of human atherosclerotic lesions and they are believed to ac... more Macrophages are prominent components of human atherosclerotic lesions and they are believed to accelerate the progression and/or complications of both early and advanced atherosclerotic lesions. We and others have shown that oxidized low-density lipoprotein (oxLDL) induces growth and inhibits apoptosis in murine bone marrow-derived macrophages. In this study, we sought to characterize the oxidative modification of LDL that is responsible for this prosurvival effect. We found that both the modified lipid and the modified protein components of oxLDL can increase the viability of macrophages. The key modification appeared to involve derivatization of amino groups in apoB or in phosphatidylethanolamine by lipid peroxidation products. These reactive oxidation products were primarily unfragmented hydroperoxide- or endoperoxide-containing oxidation products of linoleic acid or arachidonic acid. LC-MS/MS studies showed that some of the arachidonic acid-derived lysine adducts were isolevuglandins that contain lactam and hydroxylactam rings. MS/MS analysis of linoleic acid autoxidation adducts was consistent with 5- or 6-membered nitrogen-containing heterocycles derived from unfragmented oxidation products. The amine modification by oxidation products generated a fluorescence pattern with an excitation maximum at 350nm and emission maximum at 430nm. This is very similar to the fluorescence spectrum of copper-oxidized LDL.
The bioactive sphingolipid ceramide 1-phosphate (C1P) regulates cell division in a variety of cel... more The bioactive sphingolipid ceramide 1-phosphate (C1P) regulates cell division in a variety of cell types including macrophages. However, the mechanisms involved in this action are not completely understood. In the present work we show that C1P stimulates the release of vascular endothelial growth factor (VEGF) in RAW264.7 macrophages, and that this growth factor is essential for stimulation of cell proliferation by C1P. The stimulation of VEGF release was dependent upon activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB-1 also known as Akt-1), and mitogen-activated protein kinase-kinase (MEK)/extracellularly regulated kinase-2 (ERK-2) pathways, as inhibition of these kinases with selective pharmacological inhibitors or with specific gene silencing siRNA, abrogated VEGF release. A key observation was that sequestration of VEGF with a neutralizing antibody, or treatment with VEGF siRNA abolished C1P-stimulated macrophage growth. Also, inhibition of the pathways involved in C1P-stimulated VEGF release inhibited the stimulation of macrophage growth by C1P. Moreover, blockade of VEGF receptor-2 (VEGFR-2), which is the primary receptor for VEGF, with the pharmacological inhibitor DMH4, or with specific VEGFR-2 siRNA, substantially inhibited C1P-stimulated cell growth. It can be concluded that stimulation of VEGF release is a key factor in the promotion of macrophage proliferation by C1P.
International Journal of Nephrology and Renovascular Disease, Jun 1, 2022
Lipoprotein glomerulopathy (LPG) is caused by a mutation in the apolipoprotein E gene (APOE) gene... more Lipoprotein glomerulopathy (LPG) is caused by a mutation in the apolipoprotein E gene (APOE) gene and is characterized by lipoprotein thrombi in glomerular capillaries. Here, we describe a case of LPG, the first to be reported from Canada and the first case of LPG in North America to be associated with the APOE Tokyo/Maebashi mutation (p.Leu162_Lys164del, traditional nomenclature 142_144del). A 49-year-old man of Chinese descent with a previous diagnosis of dyslipidemia and a new diagnosis of hypertension was found to have proteinuria on routine urinalysis. Renal biopsy showed markedly dilated glomerular capillaries filled with pale staining mesh-like material that stained positive for Oil-RedO , consistent with lipoprotein thrombi. APOE gene sequencing confirmed the diagnosis of LPG. The patient was treated with fenofibrate and perindopril. His lipid profile normalized and proteinuria dropped to minimal levels. Repeat renal biopsy 2 years after the first showed resolution of lipoprotein thrombi but with rare residual granular densities by electron microscopy consistent with lipoprotein in the subendothelial space, supporting the hypothesis that this subendothelial material contains precursors to lipoprotein thrombi.
We tested whether loss of eukaryotic elongation factor 2 kinase (eEF2K) activity in macrophages s... more We tested whether loss of eukaryotic elongation factor 2 kinase (eEF2K) activity in macrophages suppresses development of atherosclerosis by transplanting bone marrow from mice with mutant eEF2K into ldlr Ă/Ă mice. Sixteen weeks after high-fat diet feeding, mutant eEF2K hematopoietic chimeras had a dramatically reduced level of atherosclerotic plaque formation. M1-skewed macrophages from eEF2K knock-in mice have less tumour necrosis factor-a release and a lesser ability to induce expression of endothelial cell markers, providing a potential explanation for the role of eEF2K. Because eEF2K activity in cells of the hematopoietic compartment contributes to atherosclerosis development, drugs inhibiting eEF2K might have a beneficial effect in treatment of atherosclerosis.
Clinical Journal of The American Society of Nephrology, Nov 4, 2019
Background and objectives Fibrillary GN has been defined as an immune complex-mediated GN with am... more Background and objectives Fibrillary GN has been defined as an immune complex-mediated GN with amyloidlike fibrils larger than amyloid which are IgG positive and Congo red negative. With discovery of DNAJB9 as a highly sensitive and specific marker for fibrillary GN, the specificity of the morphologic criteria for establishing the diagnosis of fibrillary GN has come into question. Design, setting, participants, & measurements We sought to (1) determine anatomic characteristics that best define fibrillary GN and (2) identify clinical and pathologic features that predict outcomes. Results We retrospectively reviewed kidney biopsies from patients diagnosed with fibrillary GN or suspected fibrillary GN between 1997 and 2017 (n=266, 65% female, median age 61). Approximately 11% of kidney biopsies had one or more unusual feature including monotypic deposits, Congo red positivity, or unusual fibril diameter. Fibrillary GN as a possible monoclonal gammopathy of renal significance represented ,1% of cases. Immunostaining for DNAJB9 confirmed fibrillary GN in 100% of cases diagnosed as fibrillary GN and 79% of atypical cases diagnosed as possible fibrillary GN. At a median time of 24 months (interquartile range, 8-46 months) after biopsy (n=100), 53% of patients reached the combined primary outcome of ESKD or death, 18% had CKD, and 18% had partial remission. On multivariable analysis, male sex (adjusted hazard ratio [aHR], 3.82; 95% confidence interval [95% CI], 1.97 to 7.37) and eGFR were the most significant predictors of primary outcome (aHR of 8.02 if eGFR ,30 ml/min per 1.73 m 2 [95% CI, 1.85 to 34.75]; aHR of 6.44 if eGFR 30 to ,45 ml/min per 1.73 m 2 [95% CI, 1.38 to 29.99]). Immunosuppressive therapy with rituximab was significantly associated with stabilization of disease progression. Conclusions Detection of DNAJB9 is a useful diagnostic tool for diagnosing atypical forms of fibrillary GN. The outcomes for fibrillary GN are poor and progression to ESKD is influenced predominantly by the degree of kidney insufficiency at the time of diagnosis and male sex. Rituximab may help preserve kidney function for select patients with fibrillary GN.
Oxidized LDL (oxLDL) promotes lipid accumulation as well as growth and survival signaling in macr... more Oxidized LDL (oxLDL) promotes lipid accumulation as well as growth and survival signaling in macrophages. OxLDL uptake is mainly due to scavenger receptors SR-AI/II and CD36. However, other scavenger receptors such as lectin-like oxLDL receptor-1 (LOX-1) may also play a role. We used mice with targeted inactivation of the LOX-1 gene to defi ne the role of this receptor in the uptake of oxLDL and in activation of survival pathways. There was no difference in uptake or degradation of 125 I-oxLDL in unstimulated macrophages from wild-type and LOX-1 knockout mice and no difference in the rate of clearance of oxLDL from plasma in vivo. However, when expression of LOX-1 was induced with lysophosphatidylcholine, oxLDL uptake and degradation increased 2-fold in wild-type macrophages but did not change in LOX-1 knockout macrophages. Macrophages lacking LOX-1 showed the same stimulation of PKB phosphorylation and enhancement of survival by oxLDL as wild-type cells. These data show that LOX-1 does not alter the uptake of oxLDL in unstimulated macrophages and is not essential for the pro-survival effect of oxLDL in these cells. However, LOX-1 expression is highly inducible by lysophosphatidylcholine and pro-infl ammatory cytokines, and if that occurred in macrophages within atheromas, LOX-1 could substantially increase oxLDL uptake by lesion macrophages.
BACKGROUND Early dysfunction of renal allografts may be associated with vascular injury, which ra... more BACKGROUND Early dysfunction of renal allografts may be associated with vascular injury, which raises the specter of active rejection processes that require medical intervention. In our practice, we have encountered patients who present with delayed graft function and demonstrate a unique pattern of endothelial cell injury that raises concern for rejection in their biopsy. Therefore, we sought to systematically determine the biopsy characteristics and outcome of these patients. METHODS During a 17-year period at the University of Washington in Seattle, United States, we identified 24 cases of a distinct arterial vasculopathy presenting in the first year posttransplantation. This early transplant arteriopathy (ETA) is characterized by endothelial cell swelling and intimal edema but without the intimal arteritis that defines vascular rejection. RESULTS Approximately 1% of transplant biopsies during the study period showed ETA, almost all of which were in deceased donor organs (96%), and most presented with delayed graft function (54%) or increased serum creatinine (38%) soon after transplantation (median 13 days; range, 5-139). In this study, 77% of patients were managed expectantly, with only 2 patients (7.6%) subsequently developing acute vascular rejection. Except for 1 patient who died, all patients had functioning allografts at 1 year follow-up. CONCLUSION Recognizing ETA and distinguishing it from vascular rejection is important to prevent over-treatment because most patients appear to recover allograft function rapidly with expectant management.
Familial hypobetalipoproteinemia (FHBL) is a rare genetic disorder of lipid metabolism that is as... more Familial hypobetalipoproteinemia (FHBL) is a rare genetic disorder of lipid metabolism that is associated with abnormally low serum levels of low-density lipoprotein (LDL) cholesterol and apolipoprotein B. It is an autosomal co-dominant disorder, and depending on zygosity, the clinical manifestations may vary from none to neurological, endocrine, hematological or liver dysfunction. Nonalcoholic fatty liver disease is common in persons with FHBL, however progression to nonalcoholic steatohepatitis is unusual. We describe here a patient with a novel APOB mutation, V703I, which appears to contribute to the severity of the FHBL phenotype. He had liver enzyme abnormalities, increased echogenicity of the liver consistent with steatosis, very low LDL cholesterol at 0.24 mmol/l (normal 1.8-3.5 mmol/l) and an extremely low apolipoprotein B level of 0.16 g/l (normal 0.6-1.2 g/l). APOB gene sequencing revealed him to be a compound heterozygote with two mutations (R463W and V703I). APOB R463W has previously been reported to cause FHBL. Genetic sequencing of his first-degree relatives identified the APOB V703I mutation in his normolipidemic brother and father and the APOB R463W mutation in his mother and sister, both of whom have very low LDL cholesterol levels. These results suggest that the APOB V703I mutation alone does not cause the FHBL phenotype. However, it is possible that it has a contributory role to a more aggressive phenotype in the presence of APOB R463W.
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