Cohort profile: the 'Biomarkers of heterogeneity in type 1 diabetes' study-a national prospective cohort study of clinical and metabolic phenotyping of individuals with long-standing type 1 diabetes in the Netherlands.

PURPOSE: The 'Biomarkers of heterogeneity in type 1 diabetes' study cohort was set up to identify genetic, physiological and psychosocial factors explaining the observed heterogeneity in disease progression and the development of complications in people with long-standing type 1 diabetes (T1D). PARTICIPANTS: Data and samples were collected in two subsets. A prospective cohort of 611 participants aged ≥16 years with ≥5 years T1D duration from four Dutch Diabetes clinics between 2016 and 2021 (median age 32 years; median diabetes duration 12 years; 59% female; mean glycated haemoglobin (HbA1c) 61 mmol/mol (7.7%); 61% on insulin pump; 23% on continuous glucose monitoring (CGM)). Physical assessments were performed, blood and urine samples were collected, and participants completed questionnaires. A subgroup of participants underwent mixed-meal tolerance tests (MMTTs) at baseline (n=169) and at 1-year follow-up (n=104). Genetic data and linkage to medical and administrative records were also available. A second cross-sectional cohort included participants with ≥35 years of T1D duration (currently n=160; median age 64 years; median diabetes duration 45 years; 45% female; mean HbA1c 58 mmol/mol (7.4%); 51% on insulin pump; 83% on CGM), recruited from five centres and measurements, samples and 5-year retrospective data were collected. FINDINGS TO DATE: Stimulated residual C-peptide was detectable in an additional 10% of individuals compared with fasting residual C-peptide secretion. MMTT measurements at 90 min and 120 min showed good concordance with the MMTT total area under the curve. An overall decrease of C-peptide at 1-year follow-up was observed. Fasting residual C-peptide secretion is associated with a decreased risk of impaired awareness of hypoglycaemia. FUTURE PLANS: Research groups are invited to consider the use of these data and the sample collection. Future work will include additional hormones, beta-cell-directed autoimmunity, specific immune markers, microRNAs, metabolomics and gene expression data, combined with glucometrics, anthropometric and clinical data, and additional markers of residual beta-cell function. TRIAL REGISTRATION NUMBER: NCT04977635.

Impact of Thyroglobulin and Thyroglobulin Antibody Assay Performance on the Differential Classification of DTC Patients.

CONTEXT: Measurements of thyroglobulin (Tg) and Tg antibodies are crucial in the follow-up of treated differentiated thyroid cancer (DTC) patients. Interassay differences may significantly impact follow-up. OBJECTIVE: The aim of this multicenter study was to explore the impact of Tg and Tg antibody assay performance on the differential classification of DTC patients, as described in national and international guidelines. DESIGN: Four commonly used Tg and Tg antibody assays were technically compared to reflect possible effects on patients with DTC follow-up. Storage stability at different storage temperatures was also investigated for LIAISON® and Kryptor assays, as this is an underexposed topic in current literature. RESULTS: B.R.A.H.M.S. assays yield approximately 50% lower Tg values over the whole range compared to the DiaSorin and Roche assays investigated. These differences between assays may result in potential misclassification in up to 7% of patients if fixed cutoffs (eg, 1 ng/mL) are applied. Poor correlation was also observed between the Tg antibody assays when the method-specific upper limits of normal are used as cutoffs. Storage of Tg and Tg antibodies was possible for 3 to 4 weeks at -20°C and -80°C. Calibration of the assays, however, was found to be crucial for stable results over time. CONCLUSIONS: Technical aspects of Tg and Tg antibody assays, including interassay differences, calibration and standardization, and cutoff values, may have a significant clinical impact on the follow-up of DTC patients.

Residual C-peptide secretion and hypoglycemia awareness in people with type 1 diabetes.

INTRODUCTION: This study aimed to assess the association between fasting serum C-peptide levels and the presence of impaired awareness of hypoglycemia (IAH) in people with type 1 diabetes. RESEARCH DESIGN AND METHODS: We performed a cross-sectional study among 509 individuals with type 1 diabetes (diabetes duration 5-65 years). Extensive clinical data and fasting serum C-peptide concentrations were collected and related to the presence or absence of IAH, which was evaluated using the validated Dutch version of the Clarke questionnaire. A multivariable logistic regression model was constructed to investigate the association of C-peptide and other clinical variables with IAH. RESULTS: In 129 (25%) individuals, residual C-peptide secretion was detected, while 75 (15%) individuals reported IAH. The median (IQR) C-peptide concentration among all participants was 0.0 (0.0-3.9) pmol/L. The prevalence of severe hypoglycemia was lower in people with demonstrable C-peptide versus those with absent C-peptide (30% vs 41%, p=0.025). Individuals with IAH were older, had longer diabetes duration, more frequently had macrovascular and microvascular complications, and more often used antihypertensive drugs, antiplatelet agents and cholesterol-lowering medication. There was a strong association between IAH and having a severe hypoglycemia in the preceding year. In multivariable regression analysis, residual C-peptide, either continuously or dichotomous, was associated with lower prevalence of IAH (p=0.040-0.042), while age at diabetes onset (p=0.001), presence of microvascular complications (p=0.003) and body mass index (BMI) (p=0.003) were also independently associated with the presence of IAH. CONCLUSIONS: Higher BMI, the presence of microvascular complications and higher age at diabetes onset were independent risk factors for IAH in people with type 1 diabetes, while residual C-peptide secretion was associated with lower risk of this complication.

Urine Concentrating Capacity, Vasopressin and Copeptin in ADPKD and IgA Nephropathy Patients with Renal Impairment.

BACKGROUND: Autosomal Dominant Polycystic Kidney Disease (ADPKD) patients have an impaired urine concentrating capacity. Increased circulating vasopressin (AVP) concentrations are supposed to play a role in the progression of ADPKD. We hypothesized that ADPKD patients have a more severely impaired urine concentrating capacity in comparison to other patients with chronic kidney disease at a similar level of kidney function, with consequently an enhanced AVP response to water deprivation with higher circulating AVP concentrations. METHODS: 15 ADPKD (eGFR<60) patients and 15 age-, sex- and eGFR-matched controls with IgA nephropathy (IgAN), underwent a water deprivation test to determine maximal urine concentrating capacity. Plasma and urine osmolality, urine aquaporin-2 (AQP2) and plasma AVP and copeptin (a surrogate marker for AVP) were measured at baseline and after water deprivation (average 16 hours). In ADPKD patients, height adjusted total kidney volume (hTKV) was measured by MRI. RESULTS: Maximal achieved urine concentration was lower in ADPKD compared to IgAN controls (533±138 vs. 642±148 mOsm/kg, p = 0.046), with particularly a lower maximal achieved urine urea concentration (223±74 vs. 299±72 mmol/L, p = 0.008). After water deprivation, plasma osmolality was similar in both groups although change in plasma osmolality was more profound in ADPKD due to a lower baseline plasma osmolality in comparison to IgAN controls. Copeptin and AVP increased significantly in a similar way in both groups. AVP, copeptin and urine AQP2 were inversely associated with maximal urine concentrating in both groups. CONCLUSIONS: ADPKD patients have a more severely impaired maximal urine concentrating capacity with a lower maximal achieved urine urea concentration in comparison to IgAN controls with similar endogenous copeptin and AVP responses.

Comparison of ex vivo stability of copeptin and vasopressin.

BACKGROUND: Copeptin, part of the vasopressin precursor, is increasingly used as marker for vasopressin and is claimed to have better ex vivo stability. However, no study has directly compared the ex vivo stability of copeptin and vasopressin. METHODS: Blood of ten healthy volunteers was collected in EDTA tubes. Next, we studied the effect of various pre-analytical conditions on measured vasopressin and copeptin levels: centrifugation speed, short-term storage temperature and differences between whole blood and plasma, long-term storage temperature and repeated freezing and thawing. The acceptable change limit (ACL), indicating the maximal percentage change that can be explained by assay variability, was used as cut-off to determine changes in vasopressin and copeptin. RESULTS: The ACL was 25% for vasopressin and 19% for copeptin. Higher centrifugation speed resulted in lower vasopressin levels, whereas copeptin concentration was unaffected. In whole blood, vasopressin was stable up to 2 h at 25°C and 6 h at 4°C. In plasma, vasopressin was stable up to 6 h at 25°C and 24 h at 4°C. In contrast, copeptin was stable in whole blood and plasma for at least 24h at both temperatures. At -20°C, vasopressin was stable up to 1 month and copeptin for at least 4 months. Both vasopressin and copeptin were stable after 4 months when stored at -80°C and -150°C. Vasopressin concentration decreased after four freeze-thaw cycles, whereas copeptin concentration was unaffected. CONCLUSION: Vasopressin levels were considerably affected by pre-analytical conditions, while copeptin levels were stable. Therefore, a strict sample handling protocol for measurement of vasopressin is recommended.

[Porphyria cutanea tarda: the benefit of additional diagnostics]. / Porphyria cutanea tarda: Nut van aanvullende diagnostiek.

The porphyrias are a clinically and genetically heterogeneous group of relatively rare metabolic diseases that result from disorders in the biosynthesis of haeme. Porphyria cutanea tarda (PCT) is the most common type, accounting for 80-90% of all porphyrias, and is essentially an acquired disease, although PCT can also occur on a familial basis. We describe a 71-year-old female and a 62-year-old male patient, both of whom had several risk factors for developing PCT, ranging from iron overload due to a mutation in the hereditary haemochromatosis protein (HFE) gene, alcohol use, smoking, and exogenous oestrogen, to persistent hepatitis C infection. The clinical relevance of the several diagnostic modalities is important in PCT. Diagnostic evaluation is important in order to confirm the diagnosis, but also to evaluate the treatment response in the context of long-term follow-up in the prevention of late complications of PCT, i.e. hepatocellular carcinoma.

Multicenter comparison study of current methods to measure 25-hydroxyvitamin D in serum.

OBJECTIVES: Measurement of serum 25-hydroxyvitamin D [25(OH)D] is generally considered to be a reliable indicator of vitamin D status. The recent increase in diversity of 25(OH)D assays prompted us to evaluate the performance of chromatographic methods (two in-house ID-LC-MS/MS and HPLC (ClinRep, Recipe)), a protein binding method (Cobas-25(OH)D-total, Roche) and immunochemical methods (Liaison and RIA (Diasorin), iSYS (IDS), ADVIA Centaur (Siemens), and Architect i1000 and i2000 (Abbott)). METHODS: Blood was drawn from randomly selected outpatients (N=60) at one site after informed consent. DEQAS and SRM 972 samples were obtained from the scheme organizer and NIST, respectively. Serum aliquots were prepared, frozen and transported to participating centers. Method comparison was performed according to CLSI-EP9 specifications. RESULTS: With these patient samples, and in comparison with ID-LC-MS/MS, Deming regression parameters slope, intercept and R were found to be within the ranges [0.57-1.07], [-1.7 to 6.9 nmol/L] and [0.88-0.98], respectively. 25(OH)D2 in DEQAS and SRM samples was fully recognized by chromatographic methods, but only partially by protein binding and immunochemical methods. Chromatographic methods, and to a lesser extent the protein binding assay, showed cross-reactivity with 3-epi-25(OH)D3. Agreement of 25(OH)D assays to ID-LC-MS/MS in sorting patients into distinct 25(OH)D categories varied between 53% and 88%. CONCLUSIONS: Significant bias exists between ID-LC-MS/MS and many, but not all, other 25(OH)D assays. The variable response among different assays for 25(OH)D metabolites impedes the use of uniform cut-off values for defining vitamin D status. Our results indicate the need towards further standardizing assays for 25(OH)D measurement.

A case of consistent discrepancies between urine and blood human chorionic gonadotropin measurements.

BACKGROUND: Our laboratory was confronted with two successive urine samples from a single patient which tested positive for human chorionic gonadotropin (hCG) when tested with both qualitative and quantitative assays, combined with no detectable hCG in corresponding plasma samples. METHODS: Serial dilution and recovery experiments were performed in order to investigate the presence of interfering substances or a high-dose hook effect. The ovarian cysts that were removed from this patient were immunohistochemically stained using polyclonal anti-human hCG antibodies. Furthermore, a urine sample was sent to the USA hCG Reference Service for hCG variant analysis. RESULTS: Dilution and recovery experiments in urine and plasma samples were unremarkable. The biopsy stained negative for human hCG and free ß-subunit. hCG isoform analysis in the urine sample revealed that approximately 87.5% of the immunoreactive hCG lacked the ß-subunit C-terminal peptide (CTP). CONCLUSIONS: We report a rare case in which two successive urine samples test positive for hCG whereas in corresponding plasma samples hCG is undetectable. The majority of the total hCG contained a degraded form of ß-subunit that lacks the CTP. This hCG variant, possibly of pituitary origin, is thought to have an extreme fast clearance rate possibly explaining the discordance between the hCG results in urine and plasma samples.

[The diagnostic value of NT-proBNP in heart failure: in depth analysis]. / De diagnostische waarde van NT-proBNP bij hartfalen.

A 77-year-old man with dyspnoea was suspected to have a decompensatio cordis by the general practitioner. A diuretic was prescribed. Additional radiological and laboratory investigation (e.g. natriuretic peptides and D-dimers) showed pulmonary embolism instead of heart failure. A second patient, a woman aged 79 years, with a history of leukaemic mantle cell lymphoma, was treated with poly-chemotherapy (R-CHOP), after which remission was achieved. Four years later the lymphoma recurred and R-CHOP treatment was started. However this was without success, after which R-CHOP treatment was repeated. Subsequently the patient developed dyspnoea and pneumonia. Following additional radiological and laboratory investigation (e.g. natriuretic peptides) the patient was finally diagnosed with doxorubicin-induced heart failure. Based upon these case studies, the role of brain-natriuretic peptides in the differential diagnostic work-up of dyspnoea is highlighted. Test performance, correlation with disease, monitoring, prognostics, differential diagnostic power, reference values and pitfalls of brain natriuretic peptides are discussed.

Macrophage p53 controls macrophage death in atherosclerotic lesions of apolipoprotein E deficient mice.

The cellular composition of atherosclerotic lesions is determined by many factors including cell infiltration, proliferation and cell death. Tumor suppressor gene p53 has been shown to regulate both cell proliferation and cell death in many cell types. In the present study, we investigated the role of macrophage p53 in the pathogenesis of early and advanced atherosclerosis. Using the Cre-loxP system we found that absence of macrophage p53 (p53(del)) strongly reduces apoptosis of macrophages both in early and advanced atherosclerotic lesions (-59% and -37%, respectively). Consequently, in advanced atherosclerosis, reduced apoptosis upon absence of macrophage p53, coincided with increased acellular necrotic core formation (+96%), increased macrophage content (+24%), and reduced cholesterol cleft accumulation (-41%). Proliferation was not affected by the absence of macrophage p53 in both early and advanced atherosclerosis. However, these significant changes in lesional cell death did not affect total lesion area in both early and advanced atherosclerosis, neither in the aortic root nor in the aortic arch and thoracic aorta in ApoE-deficient mice. Our data demonstrate that macrophage p53 is an important regulator of macrophage apoptosis, thereby preventing necrotic death of lesional macrophages. The regulation of this cell death balance directly affects lesion composition.

Dual PPARalpha/gamma agonist tesaglitazar reduces atherosclerosis in insulin-resistant and hypercholesterolemic ApoE*3Leiden mice.

OBJECTIVE: We investigated whether the dual PPARalpha/gamma agonist tesaglitazar has anti-atherogenic effects in ApoE*3Leiden mice with reduced insulin sensitivity. METHODS AND RESULTS: ApoE*3Leiden transgenic mice were fed a high-fat (HF) insulin-resistance-inducing diet. One group received a high-cholesterol (HC) supplement (1% wt/wt; HC group). A second group received the same HC supplement along with tesaglitazar (T) 0.5 micromol/kg diet (T group). A third (control) group received a low-cholesterol (LC) supplement (0.1% wt/wt; LC group). Tesaglitazar decreased plasma cholesterol by 20% compared with the HC group; cholesterol levels were similar in the T and LC groups. Compared with the HC group, tesaglitazar caused a 92% reduction in atherosclerosis, whereas a 56% reduction was seen in the cholesterol-matched LC group. Furthermore, tesaglitazar treatment significantly reduced lesion number beyond that expected from cholesterol lowering and induced a shift to less severe lesions. Concomitantly, tesaglitazar reduced macrophage-rich and collagen areas. In addition, tesaglitazar reduced inflammatory markers, including plasma SAA levels, the number of adhering monocytes, and nuclear factor kappaB-activity in the vessel wall. CONCLUSIONS: Tesaglitazar has anti-atherosclerotic effects in the mouse model that go beyond plasma cholesterol lowering, possibly caused by a combination of altered lipoprotein profiles and anti-inflammatory vascular effects.

Macrophage retinoblastoma deficiency leads to enhanced atherosclerosis development in ApoE-deficient mice.

The cellular composition of an atherosclerotic lesion is determined by cell infiltration, proliferation, and apoptosis. The tumor suppressor gene retinoblastoma (Rb) has been shown to regulate both cell proliferation and cell death in many cell types. To study the role of macrophage Rb in the development of atherosclerosis, we used apoE-deficient mice with a macrophage-restricted deletion of Rb (Rb(del) mice) and control littermates (Rb(fl) mice). After 12 wk feeding a cholesterol-rich diet, the Rb(del) mice showed a 51% increase in atherosclerotic lesion area with a 39% increase in the relative number of advanced lesions. Atherosclerotic lesions showed a 13% decrease in relative macrophage area and a 46% increase in relative smooth muscle cell area, reflecting the more advanced state of the lesions. The increase in atherosclerosis was independent of in vitro macrophage modified lipoprotein uptake or cytokine production. Whereas macrophage-restricted Rb deletion did not affect lesional macrophage apoptosis, a clear 2.6-fold increase in lesional macrophage proliferation was observed. These studies demonstrate that macrophage Rb is a suppressing factor in the progression of atherosclerosis by reducing macrophage proliferation.

Local Cre-mediated gene recombination in vascular smooth muscle cells in mice.

Here we describe a means to conditionally modify genes at a predefined and localized region of the vasculature using a perivascular drug delivery device (PDD). A 4-hydroxytamoxifen (4-OHT)-eluting PDD was applied around the carotid or femoral artery of a mouse strain carrying both the tamoxifen-inducible and smooth muscle cell (SMC)-specific Cre-recombinase (SM-Cre-ER(T2)) transgene and a stop-floxed beta-galactosidase gene in the Rosa26 locus: the SM-CreER(T2)(ki)/rosa26 mouse. A dose and time curve of 0-10% (w/w) 4-OHT and 0-14 days application of the PDD in SM-CreER(T2)(ki)/rosa26 mice showed optimal gene recombination at 1% (w/w) 4-OHT loading at 7 days post application (carotid artery 2.4+/-1.8%; femoral artery 4.0+/-3.8% of SMCs). The unique 4-OHT-eluting PDD allowed us to achieve SMC-specific recombination in the same order of magnitude as compared to systemic tamoxifen administration. In addition, recombination was completely confined to the PDD-treated vessel wall segment. Thus, local application of a 4-OHT-eluting PDD results in vascular SMC-specific Cre-mediated recombination in SM-CreER(T2)(ki)/rosa26 mice without affecting additional SMCs.

Tumor necrosis factor-alpha plays an important role in restenosis development.

Genetic factors appear to be important in the restenotic process after percutaneous coronary intervention (PCI), as well as in inflammation, a pivotal factor in restenosis. TNFalpha, a key regulator of inflammatory responses, may exert critical influence on the development of restenosis after PCI. The GENetic DEterminants of Restenosis (GENDER) project included 3104 patients who underwent a successful PCI. Systematic genotyping for six polymorphisms in the TNFalpha gene was performed. The role of TNFalpha in restenosis was also assessed in ApoE*3-Leiden mice, TNFalpha knockout mice, and by local delivery of a TNFalpha biosynthesis inhibitor, thalidomide. The -238G-1031T haplotype of the TNFalpha gene increased clinical and angiographic risk of restenosis (P=0.02 and P=0.002, respectively). In a mouse model of reactive stenosis, arterial TNFalpha mRNA was significantly time-dependently up-regulated. Mice lacking TNFalpha or treated locally with thalidomide showed a reduction in reactive stenosis (P=0.01 and P=0.005, respectively). Clinical and preclinical data indicate that TNFalpha plays an important role in restenosis. Therefore, TNFalpha genotype may be used as a risk marker for restenosis and may contribute to individual patient screening prior to PCI in clinical practice. Inhibition of TNFalpha may be an anti-restenotic target strategy.

Histopathologic alterations following local delivery of dexamethasone to inhibit restenosis in murine arteries.

OBJECTIVE: Dexamethasone-eluting stents are currently under evaluation to prevent post-angioplasty restenosis. The efficacy and safety of dexamethasone as an anti-restenotic agent is still unclear. We assess the effect of perivascular delivery of dexamethasone on vascular pathology in a mouse model of restenosis. METHODS AND RESULTS: In this study we investigate the ability of both systemic and local dexamethasone treatment to inhibit neointima formation after cuff placement around C57BL/6 mouse femoral artery. As in the clinical situation, systemic dexamethasone treatment shows adverse side effects in animals, including weight loss. In contrast, local delivery of dexamethasone using a drug-eluting polymer cuff inhibits neointima formation and has no systemic adverse effects. Pathobiological examination of the experimental arteries, however, reveals a dose-dependent medial atrophy, a reduction in vascular smooth muscle cells and collagen content, an increase in apoptotic cell count and disruption of the internal elastic lamina. CONCLUSIONS: Our results demonstrate that although local dexamethasone delivery is effective as an inhibitor for neointima formation, it is dose-dependently associated with adverse vascular morphological changes pointing to a loss of vascular integrity.

Increased vulnerability of pre-existing atherosclerosis in ApoE-deficient mice following adenovirus-mediated Fas ligand gene transfer.

OBJECTIVE: The death receptor Fas and Fas ligand (FasL) are present in human advanced atherosclerotic plaques. The activation of the Fas/FasL pathway of apoptosis has been implicated in plaque vulnerability. In the present study, we investigated whether overexpression of FasL in pre-existing atherosclerotic lesions can induce lesion remodelling and rupture-related events. METHODS AND RESULTS: Carotid atherogenesis was initiated in apolipoprotein E-deficient mice by placement of a perivascular silastic collar. The resulting plaques were incubated transluminally with recombinant adenovirus carrying FasL (Ad-FasL, lateral) or control beta-galactosidase (Ad-LacZ, contralateral). Transfection was restricted to the smooth muscle cell-rich cap of the plaque, and FasL expression led to a three-fold increase in apoptosis in the cap one day after gene transfer. Three days after gene transfer, FasL expression led to a 38% reduction in the number of cap cells. Two weeks after Ad-FasL transfer, non-thrombotic rupture, intra-plaque haemorrhage, buried caps and iron deposits were observed in 6 out of 17 Ad-FasL-treated carotid arteries versus 0 out of 17 controls (P=0.009), indicative of enhanced plaque vulnerability. CONCLUSIONS: These data demonstrate that advanced murine plaques are sensitive to Fas/FasL-induced apoptosis, which may indicate that stimulation of this pathway could result in plaque remodelling towards a more vulnerable phenotype.

Tumor necrosis factor-alpha promotes atherosclerotic lesion progression in APOE*3-Leiden transgenic mice.

OBJECTIVE: Tumor necrosis factor-alpha (TNFalpha) is a pleiotropic cytokine exerting both inflammatory and cell death modulatory activity, and is thought to play a role in the pathogenesis of atherosclerosis. Studies in mice indicated that TNFalpha affects atherosclerosis minimally or not under conditions that allow fatty streak formation. Here, we examined the possible role of TNFalpha in advanced and complex atherosclerotic lesions. METHODS AND RESULTS: To induce atherosclerosis, TNFalpha-deficient (Tnf-/-) APOE*3-Leiden and control APOE*3-Leiden only mice were fed a cholesterol-rich diet. Comparable levels of plasma cholesterol and triglycerides and the systemic inflammatory parameters, serum amyloid A and soluble intercellular adhesion molecule-1 were found in APOE*3-LeidenTnf-/- and control mice. Although absence of TNFalpha did not affect the quantitative area of atherosclerosis, APOE*3-LeidenTnf-/- mice had a higher relative number of early lesions (46.1% vs. 21.4%) and a lower relative number of advanced lesions (53.9% vs. 78.6%, P=0.04). In addition, the advanced lesions in APOE*3-LeidenTnf-/- mice showed less necrosis (9.9+/-12.1% vs. 23.4+/-19.3% of total lesion area, P=0.04) and an increase in apoptosis (1.5+/-1.5% vs. 0.4+/-0.6% of total nuclei, P=0.03). CONCLUSIONS: Our data indicate that TNFalpha stimulates the formation of lesions towards an advanced phenotype, with more lesion necrosis and a lower incidence of apoptosis.

Hepatic low-density lipoprotein receptor-related protein deficiency in mice increases atherosclerosis independent of plasma cholesterol.

The low-density lipoprotein (LDL) receptor-related protein (LRP) has a well-established role in the hepatic removal of atherogenic apolipoprotein E (APOE)-rich remnant lipoproteins from plasma. In addition, LRP recognizes multiple distinct pro- and antiatherogenic ligands in vitro. Here, we investigated the role of hepatic LRP in atherogenesis independent of its role in removal of APOE-rich remnant lipoproteins. Mice that allow inducible inactivation of hepatic LRP were combined with LDL receptor and APOE double-deficient mice (MX1Cre(+)LRP(flox/flox)LDLR(-/-)APOE(-/-)). On an LDLR(-/-)APOE(-/-) background, hepatic LRP deficiency resulted in decreased plasma cholesterol and triglycerides (cholesterol: 17.1 +/- 5.2 vs 23.4 +/- 6.3 mM, P =.025; triglycerides: 1.1 +/- 0.5 vs 2.2 +/- 0.8 mM, P =.002, for MX1Cre(+)LRP(flox/flox)-LDLR(-/-)APOE(-/-) and control LRP(flox/flox)-LDLR(-/-)APOE(-/-) mice, respectively). Lower plasma cholesterol in MX1Cre(+)LRP(flox/flox)-LDLR(-/-)APOE(-/-) mice coincided with increased plasma lipoprotein lipase (71.2 +/- 7.5 vs 19.1 +/- 2.4 ng/ml, P =.002), coagulation factor VIII (4.4 +/- 1.1 vs 1.9 +/- 0.5 U/mL, P =.001), von Willebrand factor (2.8 +/- 0.6 vs 1.4 +/- 0.3 U/mL, P =.001), and tissue-type plasminogen activator (1.7 +/- 0.7 vs 0.9 +/- 0.5 ng/ml, P =.008) compared with controls. Strikingly, MX1Cre(+)LRP(flox/flox)LDLR(-/-)APOE(-/-) mice showed a 2-fold higher atherosclerotic lesion area compared with controls (408.5 +/- 115.1 vs 219.1 +/- 86.0 10(3)microm(2), P =.003). Our data indicate that hepatic LRP plays a clear protective role in atherogenesis independent of plasma cholesterol, possibly due to maintaining low levels of its proatherogenic ligands.