Bone Marrow from Blotchy Mice Is Dispensable to Regulate Blood Copper and Aortic Pathologies but Required for Inflammatory Mediator Production in LDLR-Deficient Mice during Chronic Angiotensin II Infusion

Abstract

Background The blotchy mouse caused by mutations of ATP7A develops low blood copper and aortic aneurysm and rupture. Although the aortic pathologies are believed primarily due to congenital copper deficiencies in connective tissue, perinatal copper supplementation does not produce significant therapeutic effects, hinting additional mechanisms in the symptom development, such as an independent effect of the ATP7A mutations during adulthood. Methods We investigated if bone marrow from blotchy mice contributes to these symptoms. For these experiments, bone marrow from blotchy mice (blotchy marrow group) and healthy littermate controls (control marrow group) was used to reconstitute recipient mice (irradiated male low-density lipoprotein receptor −/− mice), which were then infused with angiotensin II (1,000 ng/kg/min) for 4 weeks. Results By using Manne–Whitney Utest, our results showed that there was no significant difference in the copper concentrations in plasma and hematopoietic cells between these 2 groups. And plasma level of triglycerides was significantly reduced in blotchy marrow group compared with that in control marrow group (P<0.05), whereas there were no significant differences in cholesterol and phospholipids between these 2 groups. Furthermore, a bead-based multiplex immunoassay showed that macrophage inflammatory protein (MIP)-1β, monocyte chemotactic protein (MCP)-1, MCP-3, MCP-5, tissue inhibitor of metalloproteinases (TIMP)-1, and vascular endothelial growth factor (VEGF)-A production was significantly reduced in the plasma of blotchy marrow group compared with that in control marrow group (P<0.05). More important, although angiotensin II infusion increased maximal external aortic diameters in thoracic and abdominal segments, there was no significant difference in the aortic diameters between these 2 groups. Furthermore, aortic ruptures, including transmural breaks of the elastic laminae in the abdominal segment and lethal rupture in the thoracic segment, were observed in blotchy marrow group but not in control marrow group; however, there was no significant difference in the incidence of aortic ruptures between these 2 groups (P = 0.10; Fisher's exact test). Conclusions Overall, our study indicated that the effect of bone marrow from blotchy mice during adulthood is dispensable in the regulation of blood copper, plasma cholesterol and phospholipids levels, and aortic pathologies, but contributes to a reduction of MIP-1β, MCP-1, MCP-3, MCP-5, TIMP-1, and VEGF-A production and triglycerides concentration in plasma. Our study also hints that bone marrow transplantation cannot serve as an independent treatment option.

Counteract of bone marrow of blotchy mice against the increases of plasma copper levels induced by high-fat diets in LDLR−/− mice

“…Moreover, transplantation of blotchy marrow followed by high-fat diets leads to a decrease in lipid profile and an increase in inflammatory mediator production. By comparing our previous study in chronic angiotensin II infused model [29], the role of blotchy marrow in copper and iron metabolism, lipid profile and inflammation was discussed here.…”

“…ATP7A-deficient female heterozygotes were crossed with GFP transgenic males to produce two new mouse strains: blotchy; GFP and wild type (WT); GFP mice [29]. A normal chow diet and an atherogenic high-fat diet (TD.88137) were used (Table 1).…”

“…Polymerase chain reaction (PCR) was employed for the genotyping of LDLR −/− mice and GFP transgenic mice, whereas PCR followed by restriction enzyme (AvaII, New England Biolabs, Ipswich, MA) digestion was conducted for the genotyping of blotchy mice and mice reconstituted with blotchy marrow as described [29]. …”

“…The mice were kept on antibiotic water for 4 wks post-radiation. There was no significant difference in the engraftment efficiency of donor cells (>90%) between the two groups of recipient mice, which was determined by measuring the percentage of GFP+ cells in the peritoneal exudates of recipient mice using flow cytometry as described [29]. …”

“…Because blotchy mice develop aortic aneurysm and rupture [28], we recently studied the contribution of blotchy marrow to aortic pathologies in a well-accepted model of aortic aneurysm and dissection (chronic infusion of angiotensin II) [29]. This study revealed that blotchy marrow has less influence on plasma copper and aortic aneurysm and dissection, although there was an increased tendency of aortic rupture.…”