Hematopoietic upstream stimulating factor 1 deficiency is associated with increased atherosclerosis susceptibility in LDL receptor knockout mice

Total body upstream stimulatory factor 1 (USF1) deficiency in mice is associated with brown adipose tissue activation and a marked protection against the development of obesity and atherosclerotic lesions. Functional expression of USF1 has also been detected in monocytes and monocyte-derived macrophages. In the current study we therefore tested whether selective hematopoietic USF1 deficiency can also beneficially impact the development of atherosclerosis. For this purpose, LDL receptor knockout mice were transplanted with bone marrow from USF1 knockout mice or their wild-type littermate controls and subsequently fed a Western-type diet for 20 weeks to stimulate atherosclerotic lesion development. Strikingly, absence of USF1 function in bone marrow-derived cells was associated with exacerbated blood leukocyte (+ 100%; P < 0.01) and peritoneal leukocyte (+ 50%; P < 0.05) lipid loading and an increased atherosclerosis susceptibility (+ 31%; P < 0.05). These effects could be attributed to aggravated hyperlipidemia, i.e. higher plasma free cholesterol (+ 33%; P < 0.001) and cholesteryl esters (+ 39%; P < 0.001), and the development of hepatosteatosis. In conclusion, we have shown that hematopoietic USF1 deficiency is associated with an increased atherosclerosis susceptibility in LDL receptor knockout mice. These findings argue against a contribution of macrophage-specific USF1 deficiency to the previously described beneficial effect of total body USF1 deficiency on atherosclerosis susceptibility in mice.Peer reviewe

Mesenchymal Stem Cells Reduce Murine Atherosclerosis Development

Mesenchymal stem cells (MSCs) have regenerative properties, but recently they were also found to have immunomodulatory capacities. We therefore investigated whether MSCs could reduce atherosclerosis, which is determined by dyslipidaemia and chronic inflammation. We adoptively transferred MSCs into low-density lipoprotein-receptor knockout mice and put these on a Western-type diet to induce atherosclerosis. Initially after treatment, we found higher levels of circulating regulatory T cells. In the long-term, overall numbers of effector T cells were reduced by MSC treatment. Moreover, MSC-treated mice displayed a significant 33% reduction in circulating monocytes and a 77% reduction of serum CCL2 levels. Most strikingly, we found a previously unappreciated effect on lipid metabolism. Serum cholesterol was reduced by 33%, due to reduced very low-density lipoprotein levels, likely a result of reduced de novo hepatic lipogenesis as determined by a reduced expression of Stearoyl-CoA desaturase-1 and lipoprotein lipase. MSCs significantly affected lesion development, which was reduced by 33% in the aortic root. These lesions contained 56% less macrophages and showed a 61% reduction in T cell numbers. We show here for the first time that MSC treatment affects not only inflammatory responses but also significantly reduces dyslipidaemia in mice. This makes MSCs a potent candidate for atherosclerosis therapies

Adrenalectomy does not alter the size (A) or collagen content (B) of atherosclerotic lesions in APOE knockout mice.

<p>Representative neutral lipid (Oil red O) and collagen (Trichrome: blue) stainings of early (C) and advanced (D) aortic root lesions. Separate dots in figures A and B represent values for each individual mouse, while horizontal lines indicate the respective group averages.</p

Plasma postprandial triglyceride response (A), cholesterol levels (B), and lipoprotein profiles (C) in adrenalectomized (ADX) and SHAM-operated APOE knockout mice.

<p>VLDL, very low-density lipoprotein; LDL, low-denisty lipoprotein; HDL, high-density lipoprotein. Values represent means+SEM or 5 mice per group. * P<0.05, ** P<0.01, *** P<0.001.</p

Adrenalectomy in APOE knockout mice does not alter the absolute thymus weight, but increases the thymic CD4/CD8 double positive lymphocyte fraction.

<p>Values represent means+SEM of 5 (FACS data) or 10-15 (thymus weight) mice per group. * P<0.05.</p

Oxidized low-density lipoprotein-induced apoptotic dendritic cells as a novel therapy for atherosclerosis

Modulation of immune responses may form a powerful approach to treat atherosclerosis. It was shown that clearance of apoptotic cells results in tolerance induction to cleared Ags by dendritic cells (DCs); however, this seems impaired in atherosclerosis because Ag-specific tolerance is lacking. This could result, in part, from decreased emigration of DCs from atherosclerotic lesions because of the high-cholesterol environment. Nonetheless, local induction of anti-inflammatory responses by apoptotic cell clearance seems to dampen atherosclerosis, because inhibition of apoptotic cell clearance worsens atherosclerosis. In this study, we assessed whether i.v. administration of oxLDL-induced apoptotic DCs (apop(ox)-DCs) and, as a control, unpulsed apoptotic DCs could modulate atherosclerosis by inducing tolerance. Adoptive transfer of apop(ox)-DCs into low-density lipoprotein receptor knockout mice either before or during feeding of a Western-type diet resulted in increased numbers of CD103(+) tolerogenic splenic DCs, with a concomitant increase in regulatory T cells. Interestingly, both types of apoptotic DCs induced an immediate 40% decrease in Ly-6C(hi) monocyte numbers and a 50% decrease in circulating CCL2 levels, but only apop(ox)-DC treatment resulted in long-term effects on monocytes and CCL2 levels. Although initial lesion development was reduced by 40% in both treatment groups, only apop(ox)-DC treatment prevented lesion progression by 28%. Moreover, progressed lesions of apop(ox)-DC-treated mice showed a robust 45% increase in collagen content, indicating an enhanced stability of lesions. Our findings clearly show that apoptotic DC treatment significantly decreases lesion development, but only apop(ox)-DCs can positively modulate lesion progression and stability. These findings may translate into a safe treatment for patients with established cardiovascular diseases using patient-derived apop(ox)-DCs

Agonistic anti-TIGIT treatment does not reduce more advanced atherosclerosis.

<p>Agonistic anti-TIGIT treatment (n = 12) and Armenian Hamster IgG treatment (n = 11) reduces atherosclerosis development in LDLr^−/− mice fed a Western-type diet for 8 weeks in comparison with PBS treatment (n = 12). Representative cross-sections of lesion formation in the three valves area of the aortic root stained with Oil-Red-O and hematoxylin are shown and lesion size was determined (A). Sections of the aortic root were stained for collagen using Masson’s trichrome staining. The percentage of collagen relative to the lesion size was determined (B). Furthermore, relative macrophage content was determined with a Moma-2 staining and quantified (C).</p

Enhanced dendritic cell percentages and activation and decreased IL-10 expressing dendritic cells after agonistic anti-TIGIT treatment.

<p>At sacrifice, blood (A) and spleen (B) cells were isolated and stained for dendritic cells and activation markers and analyzed by flow cytometry (n = 5 per group). The effect of agonistic anti-TIGIT on IL-10 expression by DCs was determined by culturing splenocytes with increasing concentrations of agonistic anti-TIGIT (C). *<i>P</i><0.05, **<i>P</i><0.01.</p

Agonistic anti-TIGIT treatment does not reduce initial atherosclerotic lesion development.

<p>No difference in atherosclerotic lesion size between agonistic anti-TIGIT, Armenian Hamster IgG and PBS treated LDLr^−/− mice fed a Western-type diet for 4 weeks. Representative cross-sections of lesion formation in the three valves area of the aortic root stained with Oil-Red-O and hematoxylin are shown and lesion size was determined (A). Sections of the aortic root were stained for collagen using Masson’s trichrome staining. The percentage of collagen relative to the lesion size was determined (B). Furthermore, relative macrophage content was determined with a Moma-2 staining and quantified (C).</p

Agonistic anti-TIGIT strongly inhibits T cell function.

<p>Splenocytes from Western-type diet fed mice (n = 3) were cultured for 72 hours with αCD3/αCD28 in the presence or absence of agonistic anti-TIGIT (0–30 µg/ml). Activated T cells (CD4⁺CD25⁺) were determined with flow cytometry (A). Proliferation was assessed by the amount of ³H-thymidine incorporation in dividing cells and is expressed as stimulation index (B) and by the amount of IL-2 produced by the splenocytes as determined with ELISA (C). Splenocytes of PBS, Armenian Hamster IgG and agonistic anti-TIGIT-treated mice (n = 5 per group) were cultured in the presence of αCD3/αCD28 stimulation and proliferation was assessed by the amount of 3H-thymidine incorporation expressed as stimulation index (D). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p