David Manka

Deposition of Platelet RANTES Triggering Monocyte Recruitment Requires P-Selectin and Is Involved in Neointima Formation After Arterial Injury

Background—Chemokines expressed on atherosclerotic endothelium or deposited by activated platelets have been implicated in monocyte recruitment during atherogenesis and restenosis. Although the involvement of P-selectin in these processes is evident from studies in knockout mice, it has not been elucidated whether delivery of platelet chemokines requires P-selectin, thus serving as a P-selectin-dependent effector function. Methods and Results—Using immunofluorescence and laminar flow assays, we found that the deposition of the platelet-derived chemokine RANTES and monocyte arrest subsequently triggered by RANTES immobilized on inflamed endothelium are more efficient after preperfusion than after static preincubation of platelets and appear to depend on interactions of platelet but not endothelial P-selectin. This was revealed by the effects of P-selectin antibodies and comparison of P-selectin-deficient and wild-type platelets. Immunohistochemistry detected a substantial luminal expression of RANTES on neointimal lesions in wire-injured carotid arteries of apolipoprotein E (apoE)-deficient mice but not of mice with a combined deficiency in apoE and P-selectin (or platelet P-selectin). As assessed by histomorphometry, treatment of apoE-deficient mice with the RANTES receptor antagonist Met-RANTES markedly reduced neointimal plaque area and macrophage infiltration. Conclusions—Our data suggest that RANTES deposition and subsequent monocyte arrest are promoted by platelet P-selectin and involved in wire-induced intimal hyperplasia, and that blocking RANTES receptors attenuates neointima formation and macrophage infiltration. This mechanism represents an important component explaining the protection against neointimal growth in P-selectin-deficient mice and may represent a novel approach to the treatment of restenosis or atherosclerosis by the administration of chemokine receptor antagonists.

Estrogen receptor-α expression in the mammary epithelium is required for ductal and alveolar morphogenesis in mice

The estrogen receptor-α (ERα) is a critical transcription factor that regulates epithelial cell proliferation and ductal morphogenesis during postnatal mammary gland development. Tissue recombination and transplantation studies using the first generation of ERα knockout (ERKO) mice suggested that this steroid hormone receptor is required in the mammary stroma that subsequently exerts its effect on the epithelium through additional paracrine signaling events. A more detailed analysis revealed that ERKO mice produce a truncated ERα protein with detectable transactivation activity, and it is likely that this functional ERα variant has masked the biological significance of this steroid receptor in the mammary epithelium. In this article, we describe the generation a Cre-lox-based conditional knockout of the ERα gene to study the biological function of this steroid receptor in the epithelial compartment at defined stages of mammary gland development. The mouse mammary tumor virus (MMTV)-Cre-mediated, epithelial-specific ablation of exon 3 of the ERα gene in virgin mice severely impaired ductal elongation and side branching. The conditional knockout resulted in ablation of the ERα protein, and the progesterone receptor (PR), whose expression is under the control of ERα, was largely absent. The whey acidic protein (WAP)-Cre-mediated deletion of ERα during successive gestation cycles resulted in a loss of ductal side-branching and lobuloalveolar structures, ductal dilation, and decreased proliferation of alveolar progenitors. These abnormalities compromised milk production and led to malnourishment of the offspring by the second lactation. These observations suggest that ERα expression in the mammary epithelium is essential for normal ductal morphogenesis during puberty and alveologenesis during pregnancy and lactation.

Crosstalk between perivascular adipose tissue and blood vessels.

Crosstalk between cells in the blood vessel wall is vital to normal vascular function and is perturbed in diseases such as atherosclerosis and hypertension. Perivascular adipocytes reside at the adventitial border of blood vessels but until recently were virtually ignored in studies of vascular function. However, perivascular adipocytes have been demonstrated to be powerful endocrine cells capable of responding to metabolic cues and transducing signals to adjacent blood vessels. Accordingly, crosstalk between perivascular adipose tissue (PVAT) and blood vessels is now being intensely examined. Emerging evidence suggests that PVAT regulates vascular function through numerous mechanisms, but evidence to date suggests modulation of three key aspects that are the focus of this review: inflammation, vasoreactivity, and smooth muscle cell proliferation.

Single Injection of P-Selectin or P-Selectin Glycoprotein Ligand-1 Monoclonal Antibody Blocks Neointima Formation After Arterial Injury in Apolipoprotein E-Deficient Mice

Background—Emerging data suggest that P-selectin, by controlling adhesion of white blood cells, may be important in limiting the response to vascular injury. Methods and Results—We tested the hypothesis that transient inhibition of P-selectin with either anti-P-selectin monoclonal antibody (mAb) or anti-P-selectin glycoprotein ligand-1 (PSGL-1) mAb would reduce neointima formation in the setting of carotid denudation injury in atherosclerosis-prone apolipoprotein E−/− mice. Neointima formation at 28 days was reduced significantly, by 50% or 80%, by a single injection on the day of injury of 100 or 200 &mgr;g P-selectin mAb RB 40.34 and by 55% by a single injection of 100 &mgr;g PSGL-1 mAb 4RA10 (P ≤0.005). In addition, there was a significant reduction in neointimal macrophage content. Conclusions—These findings demonstrate that transient P-selectin or PSGL-1 blockade at the time of arterial injury significantly limits plaque macrophage content and neointima formation in a dose-dependent manner after carotid denudation injury in apolipoprotein E−/− mice.

Histone Deacetylase 9 Is a Negative Regulator of Adipogenic Differentiation

Differentiation of preadipocytes into mature adipocytes capable of efficiently storing lipids is an important regulatory mechanism in obesity. Here, we examined the involvement of histone deacetylases (HDACs) and histone acetyltransferases (HATs) in the regulation of adipogenesis. We find that among the various members of the HDAC and HAT families, only HDAC9 exhibited dramatic down-regulation preceding adipogenic differentiation. Preadipocytes from HDAC9 gene knock-out mice exhibited accelerated adipogenic differentiation, whereas HDAC9 overexpression in 3T3-L1 preadipocytes suppressed adipogenic differentiation, demonstrating its direct role as a negative regulator of adipogenesis. HDAC9 expression was higher in visceral as compared with subcutaneous preadipocytes, negatively correlating with their potential to undergo adipogenic differentiation in vitro. HDAC9 localized in the nucleus, and its negative regulation of adipogenesis segregates with the N-terminal nuclear targeting domain, whereas the C-terminal deacetylase domain is dispensable for this function. HDAC9 co-precipitates with USF1 and is recruited with USF1 at the E-box region of the C/EBPα gene promoter in preadipocytes. Upon induction of adipogenic differentiation, HDAC9 is down-regulated, leading to its dissociation from the USF1 complex, whereas p300 HAT is up-regulated to allow its association with USF1 and accumulation at the E-box site of the C/EBPα promoter in differentiated adipocytes. This reciprocal regulation of HDAC9 and p300 HAT in the USF1 complex is associated with increased C/EBPα expression, a master regulator of adipogenic differentiation. These findings provide new insights into mechanisms of adipogenic differentiation and document a critical regulatory role for HDAC9 in adipogenic differentiation through a deacetylase-independent mechanism.

Understanding radiation-induced vascular disease.

Most cardiovascular events occur >10 years after completing radiotherapy, so demonstrating causality has proven difficult (5). An estimated 50 million cancer survivors worldwide have been treated with radiation therapy; accordingly, clinicians must be aware of the potential cardiovascular risk and manage risk factors appropriately. Moreover, research into the mechanisms of radiation-induced vascular disease is paramount to understanding and potentially modifying the disease process. The study by Martin et al. (6) in this issue of the Journal is welcome, because it sheds new light on the pathogenesis of radiation-induced vascular disease in humans. Experimental studies in animals have firmly established a causal relationship between irradiation and vascular disease. Lethal total-body irradiation of atherosclerosis-prone mice followed by bone marrow transplantation noticeably altered lesion composition and stability (7,8). Nonlethal irradiation of atherosclerosis-prone mice did not change systemic indicators of inflammation or cholesterol levels but dramatically altered lesion composition long after treatment (9). There were no changes in the atherosclerotic lesions of “out-of-field” arteries, consistent with a local rather than systemic effect of radiation. Irradiated arteries 22 to 34 weeks after treatment were highly enriched with macrophages, which accounted for the majority of the lesion area. Also, intraplaque hemorrhage was restricted to and commonly observed in irradiated arteries. These studies, however, did not identify a molecular mechanism to explain the observations. Studies to address radiation-induced vascular disease in humans have largely been descriptive in nature. From the histological perspective, lesions in medium-sized to large vessels (>100 μm in diameter) exhibit typical features of atherosclerosis, including lipid accumulation, inflammation, and thrombosis (3). Increases in intimal thickness and connective tissue content are also prominent features (2). From the angiographic perspective, the lesions are longer than traditional atherosclerotic lesions, and the regions of maximal stenosis tend to be at the ends of the lesions (10). Treating these lesions via open surgical procedures is often problematic, due to extensive soft tissue scarring; hence, percutaneous approaches are usually preferred (5). How does a course of radiotherapy initiate a chronic vascular process that eventually leads to clinical events many years after treatment? Experimental studies in vitro and in vivo indicate that radiation therapy causes acute up-regulation of pro-inflammatory cytokines and adhesion molecules in endothelium that recruits inflammatory cells to sites of vascular injury (11). It is unlikely, however, that this acute insult per se is sufficient to produce long-term occlusive atherosclerotic disease. Thus, late effects of radiation therapy are more likely responsible. In this regard, induction of chronic oxidative stress is increasingly being implicated in radiation-induced late tissue injury (12). In addition to the rapid burst of free radicals produced acutely by ionization of water molecules, radiation increases chronic free radical production and oxidative stress in the affected tissues. Oxidative stress up-regulates numerous pathways pertinent to vascular disease, including matrix metalloproteinases, adhesion molecules, pro-inflammatory cytokines, and smooth muscle cell proliferation and apoptosis, while inactivating vasculoprotective nitric oxide. Considerable evidence suggests that the nuclear transcription factor NF-κB serves as a molecular link between oxidative stress and chronic inflammation (13). The nuclear factor-kappa B (NF-κB) family of transcription factors includes 5 members: p50, p52, p65, RelB, and c-Rel. The NF-κB is involved in numerous pathological and physiological conditions, including cellular function (i.e., proliferation, differentiation, and survival), tumorigenesis, and inflammation. Upon activation, NF-κB is released from its inhibitory association with the IκB proteins in the cytoplasm and translocates to the nucleus. In the nucleus, NF-κB binding to specific deoxyribonucleic acid response elements initiates robust transcriptional responses and reprograms cellular function. Depending upon the stimuli, NF-κB can activate distinct sets of downstream genes that mediate different outcomes (14). In the context of vascular biology, NF-κB is a master regulator of inflammation and leukocyte adhesion and synchronizes the expression of adhesion molecules, cytokines, and chemokines in endothelial cells. Importantly, NF-κB is controlled by redox regulation, making it a prime candidate to link chronic oxidative stress to activation of downstream inflammatory pathways in radiation injury. The study by Martin et al. (6) provides the first direct evidence that NF-κB is chronically up-regulated in human arteries after radiation exposure. The investigators examined paired arterial specimens from skin flaps of patients who had undergone irradiation therapy for head-and-neck cancer between 4 and 500 weeks previously. They directly compared irradiated versus nonirradiated arteries from the same patients, thereby avoiding confounding inter-patient variables. Differentially expressed genes in the irradiated arteries were detected with oligonucleotide microarrays, and the data were validated by real-time polymerase chain reaction. These data were used to detect clusters of altered gene expression, which demonstrated patterns consistent with up-regulated inflammation, coagulation, and angiogenesis. Importantly, the pattern of altered gene expression in the irradiated arteries suggested transcriptional regulation by NF-κB. Indeed, immunohistological studies demonstrated up-regulated NF-κB in vascular wall cells, with specific localization to macrophages. Interestingly, a group of putative NF-κB– dependent genes were found to be similarly dysregulated, regardless of the amount of time since irradiation (4 to 7 weeks vs. 20 to 500 weeks). A similar study with larger number of patients would be needed to separate early-stage from late-stage gene expression programs, which might be highly informative. The study by Martin et al. (6) provides the foundation for a molecular mechanism to explain the effects of radiation on vascular biology (Fig. 1). As with many studies that address a poorly understood phenomenon, the data generate more questions than answers. Are the results observed in the small conduit arteries in this study translatable to large arteries that produce most cardiovascular events? Does oxidative stress cause the persistent NF-κB up-regulation and reprogramming of gene expression in irradiated blood vessels? Lastly and most importantly, could modulation of NF-κB ameliorate the disease process? Several commonly used agents, such as aspirin, omega-3 fatty acids, and statins, directly or indirectly modulate NF-κB activity; whether these medications could ameliorate radiation-induced vascular disease remains to be determined. Also, inhibitors of NF-κB are being tested for a variety of inflammatory states and might eventually make their way into clinical medicine (13,14). Perhaps such therapy could be employed to treat radiation-induced vascular disease. Alternatively, the pathways responsible for up-regulated oxidative stress might be targeted. In this regard, activation of the angiotensin II-aldosterone system has been hypothesized to play a key role in propagating oxidative stress after radiation therapy (12,15). Thus, pharmacotherapy directed against this pathway could potentially be efficacious against radiation-induced vascular disease. Figure 1 Proposed Mechanism of Involvement of NF-κB in Radiation-Induced Vascular Disease In conclusion, Martin et al. (6) have made an important contribution to the field of radiation-induced vascular disease by demonstrating local and sustained up-regulation of NF-κB in irradiated human blood vessels. The expression profiles suggest that NF-κB contributes to the pathology by inducing pro-inflammatory genes. Further research is needed to determine the clinical significance of their findings and to investigate whether currently available and/or emerging therapies can modulate the disease process.

Human coronary artery perivascular adipocytes overexpress genes responsible for regulating vascular morphology, inflammation, and hemostasis

Inflammatory cross talk between perivascular adipose tissue and the blood vessel wall has been proposed to contribute to the pathogenesis of atherosclerosis. We previously reported that human perivascular (PV) adipocytes exhibit a proinflammatory phenotype and less adipogenic differentiation than do subcutaneous (SQ) adipocytes. To gain a global view of the genomic basis of biologic differences between PV and SQ adipocytes, we performed genome-wide expression analyses to identify differentially expressed genes between adipocytes derived from human SQ vs. PV adipose tissues. Although >90% of well-expressed genes were similarly regulated, we identified a signature of 307 differentially expressed genes that were highly enriched for functions associated with the regulation of angiogenesis, vascular morphology, inflammation, and blood clotting. Of the 156 PV upregulated genes, 59 associate with angiogenesis, vascular biology, or inflammation, noteworthy of which include TNFRSF11B (osteoprotegerin), PLAT, TGFB1, THBS2, HIF1A, GATA6, and SERPINE1. Of 166 PV downregulated genes, 21 associated with vascular biology and inflammation, including ANGPT1, ANGPTL1, and VEGFC. Consistent with the emergent hypothesis that PV adipocytes differentially regulate angiogenesis and inflammation, cell culture-derived adipocyte-conditioned media from PV adipocytes strongly enhanced endothelial cell tubulogenesis and monocyte migration compared with media from SQ adipocytes. These findings demonstrate that PV adipocytes have the potential to significantly modulate vascular inflammatory crosstalk in the setting of atherosclerosis by their ability to signal to both endothelial and inflammatory cells.

Arterial Injury Increases Expression of Inflammatory Adhesion Molecules in the Carotid Arteries of Apolipoprotein-E-Deficient Mice1

Recent studies demonstrate increased cellular adhesion molecule expression by neointimal endothelium overlying primary and restenotic atherosclerotic plaque. In this study, we developed an atherosclerotic mouse model of arterial injury and characterized adhesion molecule expression after injury. Sixteen apolipoprotein-E-(ApoE)-deficient mice fed a Western-type diet for 4 weeks underwent carotid artery wire denudation at week 2. For each segment, the extent of neointima formation and medial thickening, or adhesion molecule expression, were scored separately on a scale from 0 (no plaque/thickening or expression) to 3 (extensive plaque/thickening or expression) using Movat staining (n = 3) or immunohistochemical analysis (n = 13). Histology revealed significant medial thickening (1.8 ± 0.9 vs. 0.3 ± 0.5, p < 0.001) versus controls and pronounced staining for monocytes/macrophages in the wall of injured vessels. Immunohistochemical analysis showed more robust expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on the luminal surface of injured arteries versus controls (2.2 ± 0.6 vs. 1.4 ± 0.7, p < 0.01, and 2.5 ± 0.5 vs. 1.2 ± 0.6, p < 0.001, respectively). Injury increased adventitial ICAM-1 expression (2.6 ± 0.5 vs. 1.6 ± 0.5, p < 0.002) and medial VCAM-1 expression (2.2 ± 0.6 vs. 1.2 ± 0.7, p < 0.004). Thus, carotid injury results in significant medial thickening and increases adhesion molecule expression beyond that induced in ApoE-deficient mice fed a Western diet alone. The observation of macrophage infiltration into the media at sites of increased ICAM-1 and VCAM-1 expression suggests that these molecules may mediate monocyte/macrophage trafficking into the wall of injured arteries.

Transplanted perivascular adipose tissue accelerates injury-induced neointimal hyperplasia: role of monocyte chemoattractant protein-1.

Objective— Perivascular adipose tissue (PVAT) expands during obesity, is highly inflamed, and correlates with coronary plaque burden and increased cardiovascular risk. We tested the hypothesis that PVAT contributes to the vascular response to wire injury and investigated the underlying mechanisms. Approach and Results— We transplanted thoracic aortic PVAT from donor mice fed a high-fat diet to the carotid arteries of recipient high-fat diet–fed low-density lipoprotein receptor knockout mice. Two weeks after transplantation, wire injury was performed, and animals were euthanized 2 weeks later. Immunohistochemistry was performed to quantify adventitial macrophage infiltration and neovascularization and neointimal lesion composition and size. Transplanted PVAT accelerated neointimal hyperplasia, adventitial macrophage infiltration, and adventitial angiogenesis. The majority of neointimal cells in PVAT-transplanted animals expressed &agr;-smooth muscle actin, consistent with smooth muscle phenotype. Deletion of monocyte chemoattractant protein-1 in PVAT substantially attenuated the effects of fat transplantation on neointimal hyperplasia and adventitial angiogenesis, but not adventitial macrophage infiltration. Conditioned medium from perivascular adipocytes induced potent monocyte chemotaxis in vitro and angiogenic responses in cultured endothelial cells. Conclusions— These findings indicate that PVAT contributes to the vascular response to wire injury, in part through monocyte chemoattractant protein-1–dependent mechanisms.

Transplanted Perivascular Adipose Tissue Accelerates Injury-Induced Neointimal Hyperplasia

Objective— Perivascular adipose tissue (PVAT) expands during obesity, is highly inflamed, and correlates with coronary plaque burden and increased cardiovascular risk. We tested the hypothesis that PVAT contributes to the vascular response to wire injury and investigated the underlying mechanisms. Approach and Results— We transplanted thoracic aortic PVAT from donor mice fed a high-fat diet to the carotid arteries of recipient high-fat diet–fed low-density lipoprotein receptor knockout mice. Two weeks after transplantation, wire injury was performed, and animals were euthanized 2 weeks later. Immunohistochemistry was performed to quantify adventitial macrophage infiltration and neovascularization and neointimal lesion composition and size. Transplanted PVAT accelerated neointimal hyperplasia, adventitial macrophage infiltration, and adventitial angiogenesis. The majority of neointimal cells in PVAT-transplanted animals expressed &agr;-smooth muscle actin, consistent with smooth muscle phenotype. Deletion of monocyte chemoattractant protein-1 in PVAT substantially attenuated the effects of fat transplantation on neointimal hyperplasia and adventitial angiogenesis, but not adventitial macrophage infiltration. Conditioned medium from perivascular adipocytes induced potent monocyte chemotaxis in vitro and angiogenic responses in cultured endothelial cells. Conclusions— These findings indicate that PVAT contributes to the vascular response to wire injury, in part through monocyte chemoattractant protein-1–dependent mechanisms.