Soluble PD-1 (sPD-1) is expressed in human macrophages.

The PD-1/PD-L1 axis plays a crucial role in regulating the anti-tumour immune response. A soluble PD-1 protein (sPD-1) has previously been observed, which could block the binding of PD-L1 to PD-1. Tumour associated macrophages are abundant in tumours, and evidence suggest they express PD-1. However, whether they also express sPD-1 remains unclear. The objective of this study was to investigate expression of sPD-1 in two in vitro models of human macrophages: THP-1 cells and monocyte-derived macrophages (MDM). Cells were polarised with either LPS + IFN-γ or IL-4 + IL-13 or left unpolarised. PD-1 and sPD-1 mRNAs were measured using droplet digital PCR, sPD-1 protein by electrochemiluminescence immunoassay and PD-1 by flow cytometry. sPD-1 mRNA was induced in both THP-1 cells and MDM after polarisation with LPS + IFN-γ, while IL-4 + IL-13 induced sPD-1 mRNA in MDM only. sPD-1 protein was measurable in culture supernatants. These findings show that macrophages can be induced to express sPD-1.

Increased retention of LDL from type 1 diabetic patients in atherosclerosis-prone areas of the murine arterial wall.

BACKGROUND AND AIMS: Type 1 diabetes accelerates the development of atherosclerotic cardiovascular diseases. Retention of low-density lipoprotein (LDL) in the arterial wall is a causal step in atherogenesis, but it is unknown whether diabetes alters the propensity of LDL for retention. The present study investigated whether LDL from type 1 diabetic and healthy non-diabetic subjects differed in their ability to bind to the arterial wall in a type 1 diabetic mouse model. METHODS: Fluorescently-labeled LDL obtained from type 1 diabetic patients or healthy controls was injected into mice with type 1 diabetes. The amount of retained LDL in the atherosclerosis-prone inner curvature of the aortic arch was quantified by fluorescence microscopy. Healthy control LDL was in vitro glycated, analyzed for protein glycation by LC-MS/MS, and tested for retention propensity. RESULTS: Retention of LDL from type 1 diabetic patients was 4.35-fold higher compared to LDL from nondiabetic subjects. Nuclear magnetic resonance (NMR) spectroscopy analysis of LDL revealed no differences in the concentration of the atherogenic small dense LDL between type 1 diabetic and non-diabetic subjects. In vitro glycation of LDL from a non-diabetic subject increased retention compared to non-glycated LDL. LC-MS/MS revealed four new glycated spots in the protein sequence of ApoB of in vitro glycated LDL. CONCLUSIONS: LDL from type 1 diabetic patients showed increased retention at atherosclerosis-prone sites in the arterial wall of diabetic mice. Glycation of LDL is one modification that may increase retention, but other, yet unknown, mechanisms are also likely to contribute.

Type 1 diabetes increases retention of low-density lipoprotein in the atherosclerosis-prone area of the murine aorta.

BACKGROUND AND AIMS: Individuals with type 1 diabetes mellitus are at high risk of developing atherosclerotic cardiovascular disease, but the underlying mechanisms by which type 1 diabetes accelerates atherosclerosis remain unknown. Increased retention of low-density lipoprotein (LDL) in atherosclerosis-prone sites of the diabetic vascular wall has been suggested, but direct evidence is lacking. In the present study, we investigated whether retention of LDL is increased in atherosclerotic-prone areas using a murine model of type 1 diabetes. METHODS: Fluorescently-labeled human LDL from healthy non-diabetic individuals was injected into diabetic Ins2Akita mice and non-diabetic, wild-type littermates. The amount of retained LDL after 24 h was quantified by fluorescence microscopy of cryosections and by scans of en face preparations. Vascular gene expression in the inner curvature of the aortic arch was analyzed by microarray and quantitative polymerase chain reaction. RESULTS: LDL retention was readily detected in atherosclerosis-prone areas of the aortic arch being located in both intimal and medial layers. Quantitative microscopy revealed 8.1-fold more retained LDL in type 1 diabetic mice compared to wild-type mice. These findings were confirmed in independent experiments using near-infrared scanning of en face preparations of the aorta. Diabetic status did not affect arterial expression of genes known to be involved in LDL retention. CONCLUSIONS: Type 1 diabetes increases the ability of the vascular wall to retain LDL in mice. These changes could contribute to the increased atherosclerotic burden seen in type 1 diabetic patients.

Apolipoprotein E Deficiency Increases Remnant Lipoproteins and Accelerates Progressive Atherosclerosis, But Not Xanthoma Formation, in Gene-Modified Minipigs.

Deficiency of apolipoprotein E (APOE) causes familial dysbetalipoproteinemia in humans resulting in a higher risk of atherosclerotic disease. In mice, APOE deficiency results in a severe atherosclerosis phenotype, but it is unknown to what extent this is unique to mice. In this study, APOE was targeted in Yucatan minipigs. APOE-/- minipigs displayed increased plasma cholesterol and accumulation of apolipoprotein B-48-containing chylomicron remnants on low-fat diet, which was significantly accentuated upon feeding a high-fat, high-cholesterol diet. APOE-/- minipigs displayed accelerated progressive atherosclerosis but not xanthoma formation. This indicates that remnant lipoproteinemia does not induce early lesions but is atherogenic in pre-existing atherosclerosis.

Prior renovascular hypertension does not predispose to atherosclerosis in mice.

BACKGROUND: Hypertension is a major risk factor for development of atherosclerotic cardiovascular disease (ASCVD). Although lowering blood pressure with antihypertensive drugs reduces the increased risk of ASCVD, residual increased risk still remains, suggesting that hypertension may cause chronic changes that promote atherosclerosis. Thus, we tested the hypothesis that hypertension increases the susceptibility to atherosclerosis in mice even after a period of re-established normotension. METHODS: We used the 2-kidney, 1-clip (2K1C) technique to induce angiotensin-driven renovascular hypertension, and overexpression of the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene to cause severe hypercholesterolemia and atherosclerosis. RESULTS: First, we performed 2K1C (n = 8) or sham surgery (n = 9) in PCSK9 transgenic mice before they were fed a high fat diet for 14 weeks. As expected, 2K1C did not affect cholesterol levels, but induced cardiac hypertrophy and significantly increased the atherosclerotic lesion area compared to sham mice (1.8 fold, p < 0.01). Next, we performed 2K1C (n = 13) or sham surgery (n = 14) in wild-type mice but removed the clipped/sham-operated kidney after 10 weeks to eliminate hypertension, and subsequently induced hypercholesterolemia by way of adeno-associated virus-mediated hepatic gene transfer of PCSK9 combined with high-fat diet. After 14 weeks of hypercholesterolemia, atherosclerotic lesion areas were not significantly different in mice with or without prior 2K1C hypertension (0.95 fold, p = 0.35). CONCLUSION: Renovascular hypertension in mice does not induce pro-atherogenic changes that persist beyond the hypertensive phase. These results indicate that hypertension only promotes atherogenesis when coinciding temporally with hypercholesterolemia.

Arterial endothelial cells: still the craftsmen of regenerated endothelium.

For more than a decade, a prevailing hypothesis in research related to arterial disease has been that circulating endothelial progenitor cells (EPCs) provide protection by their innate ability to replace dysfunctional or damaged endothelium. This paradigm has led to extensive investigation of EPCs in the hope of finding therapeutic targets to control their homing and differentiation. However, from the very beginning, the nomenclature and the phenotype of EPCs have been subject to controversy and there are currently no specific markers that can unambiguously identify these cells. Moreover, many of the initial observations that EPCs differentiate to endothelial cells in the course of arterial disease have been criticized for methodological problems. The present review discusses the contrasting experimental evidence as to the role of EPCs in contributing to relining of the endothelium and highlights some of the methodological pitfalls and terminological ambiguities that confuse the field.

Circulating endothelial progenitor cells do not contribute to regeneration of endothelium after murine arterial injury.

AIMS: Endothelial regeneration after vascular injury, including percutaneous coronary intervention, is essential for vascular homeostasis and inhibition of neointima formation. Circulating endothelial progenitor cells (EPCs) have been implicated to contribute by homing and differentiating into endothelial cells (ECs). We tested this theory in a murine arterial injury model using carotid artery transplants and fluorescent reporter mice. METHODS AND RESULTS: Wire-injured carotid artery segments from wild-type mice were transplanted into TIE2-GFP transgenic mice expressing green fluorescent protein (GFP) in ECs. We found that the endothelium regenerated with GFP(+) ECs as a function of time, evolving from the anastomosis sites towards the centre of the transplant. A migration front of ECs at Day 7 was verified by scanning electron microscopy and by bright-field microscopy using recipient TIE2-lacZ mice with endothelial ß-galactosidase expression. These experiments indicated migration of flanking ECs rather than homing of circulating cells as the underlying mechanism. To confirm this, we interposed non-injured wild-type carotid artery segments between the denuded transplant and the TIE2-GFP recipient mouse. Among 1186 ECs identified in re-endothelialized transplants (n= 5) by staining for von Willebrand Factor or vascular endothelial-cadherin, we did not find any blood-derived (GFP(+)) cells. CONCLUSION: Endothelial regeneration after vascular injury did not involve circulating EPCs but was mediated solely by migration of ECs from the adjacent healthy endothelium.

Flanking recipient vasculature, not circulating progenitor cells, contributes to endothelium and smooth muscle in murine allograft vasculopathy.

OBJECTIVE: The prevailing view assumes that circulating endothelial and smooth muscle progenitor cells participate in allograft vasculopathy (AV), although the seminal studies in the field were not designed to distinguish between circulating and migrating cells of recipient origin. We developed a double-transplantation technique to overcome this problem and reinvestigated the origin of endothelial cells (ECs) and smooth muscle cells (SMCs) in murine AV. METHODS AND RESULTS: Carotid artery segments from BALB/c mice were allografted to apolipoprotein E(-/-) B6 mice with or without a "flanking" isograft interpositioned between the allograft and the recipient artery. Either recipient mice or interpositioned isografts expressed enhanced green fluorescent protein, and consequently, cells migrating into the allograft from the flanking vasculature could easily be tracked and distinguished from recruited circulating cells. Without immunosuppression, allograft donor cells vanished as expected, and AV developed by replacement and accumulation of ECs and SMCs of recipient origin. The double transplantation models revealed that all ECs and SMCs in AV had migrated into the allograft from the flanking vasculature without any contribution from putative progenitor cells in the blood. CONCLUSIONS: Migrating cells from the flanking vasculature, not circulating progenitor cells, are the source of recipient-derived ECs and SMCs in murine AV.

Baroreflex control of heart rate in the broad-nosed caiman Caiman latirostris is temperature dependent.

It has been suggested that ectothermic vertebrates primarily control blood pressure to protect the pulmonary vasculature from oedema caused by high pressure, while endothermic vertebrates control blood pressure to maintain adequate oxygen delivery to the tissues. In the present study we have characterised how temperature affects the cardiac limb of the baroreflex in the intact unanaesthetized broad-nosed caiman (Caiman latirostris) by pharmacological manipulation of blood pressure in a "closed-loop" system. Sodium nitroprusside (SNP) and phenylephrine were used to manipulate arterial pressure and the resulting alterations in heart rate were used to calculate the gain of the baroreflex. Both drugs were infused as bolus injections in concentrations of 5, 10, 25, 50 and 100 microg kg(-1). The barostatic response was present at both 15 and 30 degrees C, and, at both temperatures, C.latirostris responded to reductions in systemic blood pressure (Psys). At 30 degrees C the baroreflex was more pronounced at a blood pressure lower than control value (52.3 cmH(2)O) with a maximal baroreflex gain of 1.97 beats min(-)(1)cmH(2)O(-1) at a Psys of 41.9 cmH(2)O, and therefore seems to counteract hypotension. In contrast, the maximal baroreflex at 15 degrees C was found at a Psys almost equal to the control value. The highest baroreflex gain in response to change in blood pressure was measured at the highest temperature. Thus, C.latirostris exhibit a temperature dependent barostatic response.

Circulating endothelial progenitor cells do not contribute to plaque endothelium in murine atherosclerosis.

BACKGROUND: It has been reported that circulating endothelial progenitor cells (EPCs) home to and differentiate into endothelial cells after various kinds of arterial injury. By inference, EPCs are also proposed to be important in the most important arterial disease, atherosclerosis, but the evidence for this theory is not clear. In the present study, we assessed the contribution of circulating EPCs to plaque endothelium in apolipoprotein E-deficient (apoE(-/-)) mice. METHODS AND RESULTS: To investigate whether EPCs in the circulating blood are a source of plaque endothelial cells during atherogenesis, we examined plaques in lethally irradiated apoE(-/-) mice reconstituted with bone marrow cells from enhanced green fluorescent protein (eGFP) transgenic apoE(-/-) mice and plaques induced in segments of common carotid artery transplanted from apoE(-/-) mice into eGFP(+)apoE(-/-) mice. Among 4232 endothelial cells identified by a cell-type-specific marker (von Willebrand factor) and analyzed by high-resolution microscopy, we found only 1 eGFP(+). Using the Y chromosome to track cells after sex-mismatched transplants yielded similar results. To investigate whether circulating EPCs are involved in plaque reendothelialization after plaque disruption and superimposed thrombosis, we produced mechanical plaque disruptions in carotid bifurcation plaques in old lethally irradiated apoE(-/-) mice reconstituted with eGFP(+)apoE(-/-) bone marrow cells and carotid bifurcation plaques transplanted from old apoE(-/-) mice into eGFP(+)apoE(-/-) mice. Only 1 eGFP(+) endothelial cell was found among 3170 analyzed. CONCLUSIONS: Circulating EPCs rarely, if ever, contribute to plaque endothelium in apoE(-/-) mice. These findings bring into question the prevailing theory that circulating EPCs play an important role in atherogenesis.

Physiological importance of the coronary arterial blood supply to the rattlesnake heart.

The reptilian heart consists of a thick inner spongy myocardium that derives its oxygen and nutrient supply directly from the blood within the ventricular cavity, which is surrounded by a thin outer compact layer supplied by coronary arteries. The functional importance of these coronary arteries remains unknown. In the present study we investigate the effects of permanent coronary artery occlusion in the South American rattlesnake (Crotalus durissus) on the ability to maintain heart rate and blood pressure at rest and during short term activity. We used colored silicone rubber (Microfil) to identify the coronary artery distribution and interarterial anastomoses. The coronary circulation was occluded and the snakes were then kept for 4 days at 30 degrees C. Microfil injections verified that virtually all coronary arteries had successfully been occluded, but also made visible an extensive coronary supply to the outer compact layer in untreated snakes. Electrocardiogram (ECG), blood pressure (Psys) and heart rate (fH) were measured at rest and during enforced activity at day 1 and 4. Four days after occlusion of the coronary circulation, the snakes could still maintain a Psys and fH of 5.2+/-0.2 kPa and 58.2+/-2.2 beats min(-1), respectively, during activity and the ECG was not affected. This was not different from sham-operated snakes. Thus, while the outer compact layer of the rattlesnake heart clearly has an extensive coronary supply, rattlesnakes sustain a high blood pressure and heart rate during activity without coronary artery blood supply.