Influence of hypoxia on the domiciliation of Mesenchymal Stem Cells after infusion into rats: possibilities of targeting pulmonary artery remodeling via cells therapies?

Background

Bone marrow (BM) cells are promising tools for vascular therapies. Here, we focused on the possibility of targeting the hypoxia-induced pulmonary artery hypertension remodeling with systemic delivery of BM-derived mesenchymal stem cells (MSCs) into non-irradiated rats.

Methods

Six-week-old Wistar rats were exposed to 3-week chronic hypoxia leading to pulmonary artery wall remodeling. Domiciliation of adhesive BM-derived CD45^- CD73⁺ CD90⁺ MSCs was first studied after a single intravenous infusion of Indium-111-labeled MSCs followed by whole body scintigraphies and autoradiographies of different harvested organs. In a second set of experiments, enhanced-GFP labeling allowed to observe distribution at later times using sequential infusions during the 3-week hypoxia exposure.

Results

A 30% pulmonary retention was observed by scintigraphies and no differences were observed in the global repartition between hypoxic and control groups. Intrapulmonary radioactivity repartition was homogenous in both groups, as shown by autoradiographies. BM-derived GFP-labeled MSCs were observed with a global repartition in liver, in spleen, in lung parenchyma and rarely in the adventitial layer of remodeled vessels. Furthermore this global repartition was not modified by hypoxia. Interestingly, these cells displayed in vivo bone marrow homing, proving a preservation of their viability and function. Bone marrow homing of GFP-labeled MSCs was increased in the hypoxic group.

Conclusion

Adhesive BM-derived CD45^- CD73⁺ CD90⁺ MSCs are not integrated in the pulmonary arteries remodeled media after repeated intravenous infusions in contrast to previously described in systemic vascular remodeling or with endothelial progenitor cells infusions.     

Hypoxia, leukocytes, and the pulmonary circulation.

Data are rapidly accumulating in support of the idea that circulating monocytes and/or mononuclear fibrocytes are recruited to the pulmonary circulation of chronically hypoxic animals and that these cells play an important role in the pulmonary hypertensive process. Hypoxic induction of monocyte chemoattractant protein-1, stromal cell-derived factor-1, vascular endothelial growth factor-A, endothelin-1, and tumor growth factor-beta(1) in pulmonary vessel wall cells, either directly or indirectly via signals from hypoxic lung epithelial cells, may be a critical first step in the recruitment of circulating leukocytes to the pulmonary circulation. In addition, hypoxic stress appears to induce release of increased numbers of monocytic progenitor cells from the bone marrow, and these cells may have upregulated expression of receptors for the chemokines produced by the lung circulation, which thus facilitates their specific recruitment to the pulmonary site. Once present, macrophages/fibrocytes may exert paracrine effects on resident pulmonary vessel wall cells stimulating proliferation, phenotypic modulation, and migration of resident fibroblasts and smooth muscle cells. They may also contribute directly to the remodeling process through increased production of collagen and/or differentiation into myofibroblasts. In addition, they could play a critical role in initiating and/or supporting neovascularization of the pulmonary artery vasa vasorum. The expanded vasa network may then act as a conduit for further delivery of circulating mononuclear cells to the pulmonary arterial wall, creating a feedforward loop of pathological remodeling. Future studies will need to determine the mechanisms that selectively induce leukocyte/fibrocyte recruitment to the lung circulation under hypoxic conditions, their direct role in the remodeling process via production of extracellular matrix and/or differentiation into myofibroblasts, their impact on the phenotype of resident smooth muscle cells and adventitial fibroblasts, and their role in the neovascularization observed in hypoxic pulmonary hypertension.     

Notch Signaling Change in Pulmonary Vascular Remodeling in Rats with Pulmonary Hypertension and Its Implication for Therapeutic Intervention

Pulmonary hypertension (PH) is a fatal disease that lacks an effective therapy. Notch signaling pathway plays a crucial role in the angiogenesis and vascular remodeling. However, its roles in vascular remodeling in PH have not been well studied. In the current study, using hypoxia-induced PH model in rat, we examined the expression of Notch and its downstream factors. Then, we used vessel strip culture system and γ-secretase inhibitor DAPT, a Notch signaling inhibitor to determine the effect of Notch signaling in vascular remodeling and its potential therapeutic value. Our results indicated that Notch 1–4 were detected in the lung tissue with variable levels in different cell types such as smooth muscle cells and endothelial cells of pulmonary artery, bronchia, and alveoli. In addition, following the PH induction, all of Notch1, Notch3, Notch4 receptor, and downstream factor, HERP1 in pulmonary arteries, mRNA expressions were increased with a peak at 1–2 weeks. Furthermore, the vessel wall thickness from rats with hypoxia treatment increased after cultured for 8 days, which could be decreased approximately 30% by DAPT, accompanied with significant increase of expression level of apoptotic factors (caspase-3 and Bax) and transformation of vascular smooth muscle cell (VSMC) phenotype from synthetic towards contractile. In conclusion, the current study suggested Notch pathway plays an important role in pulmonary vascular remodeling in PH and targeting Notch signaling pathway could be a valuable approach to design new therapy for PH.     

慢性低氧所致肺动脉高压对大鼠肺血管平滑肌细胞及成纤维细胞中蛋白激酶CβI的膜转位和蛋白表达量的影响

目的通过观察慢性低氧所致肺动脉高压对大鼠肺血管平滑肌细胞及成纤维细胞中蛋白激酶CBI（PKCβI）的膜转位和蛋白表达量的影响,初步探讨PKCpI在慢性低氧诱导大鼠肺动脉高压的发生、发展过程中所起的作用。方法建立慢性常压低氧肺动脉高压大鼠模型,将雄性SD大鼠随机分为正常对照组、低氧1d、3d、7d、14d和21d组,应用蛋白免疫印迹和免疫组化技术检测肺动脉高压形成过程中大鼠肺血管平滑肌细胞及成纤维细胞中PKCβI的膜转位和蛋白表达水平。结果（1）RVSP和RV／（LV＋S）比值较正常对照组明显增加（P〈0．05）,低氧后3d、7d、14d和21d后大鼠肺血管明显增厚;（2）大鼠肺血管平滑肌细胞和成纤维细胞均有PKCβI的表达,且低氧14d后PKCβI的蛋白表达量较正常对照组相比降低（P〈0．05）。结论PKCβI蛋白表达量的下调可能参与了慢性低氧诱导的大鼠肺动脉高压肺血管重塑的发生、发展过程。     

miR-21 regulates chronic hypoxia-induced pulmonary vascular remodeling

Chronic hypoxia causes pulmonary vascular remodeling leading to pulmonary hypertension (PH) and right ventricle (RV) hypertrophy. Aberrant expression of microRNA (miRNA) is closely associated with a number of pathophysiologic processes. However, the role of miRNAs in chronic hypoxia-induced pulmonary vascular remodeling and PH has not been well characterized. In this study, we found increased expression of miR-21 in distal small arteries in the lungs of hypoxia-exposed mice. Putative miR-21 targets, including bone morphogenetic protein receptor (BMPR2), WWP1, SATB1, and YOD1, were downregulated in the lungs of hypoxia-exposed mice and in human pulmonary artery smooth muscle cells (PASMCs) overexpressing miR-21. We found that sequestration of miR-21, either before or after hypoxia exposure, diminished chronic hypoxia-induced PH and attenuated hypoxia-induced pulmonary vascular remodeling, likely through relieving the suppressed expression of miR-21 targets in the lungs of hypoxia-exposed mice. Overexpression of miR-21 enhanced, whereas downregulation of miR-21 diminished, the proliferation of human PASMCs in vitro and the expression of cell proliferation associated proteins, such as proliferating cell nuclear antigen, cyclin D1, and Bcl-xL. Our data suggest that miR-21 plays an important role in the pathogenesis of chronic hypoxia-induced pulmonary vascular remodeling and also suggest that miR-21 is a potential target for novel therapeutics to treat chronic hypoxia associated pulmonary diseases.     

Vasa vasorum inside out/outside in communication: a potential role in the patency of saphenous vein coronary artery bypass grafts

The saphenous vein (SV) is the most commonly used conduit for revascularization in patients undergoing coronary artery bypass surgery (CABG). The patency rate of this vessel is inferior to the internal thoracic artery (ITA). In the majority of CABG procedures the ITA is removed with its outer pedicle intact whereas the (human) SV (hSV) is harvested with pedicle removed. The vasa vasorum, a microvessel network providing the adventitia and media with oxygen and nutrients, is more pronounced and penetrates deeper towards the lumen in veins than in arteries. When prepared in conventional CABG the vascular trauma caused when removing the hSV pedicle damages the vasa vasorum, a situation affecting transmural flow potentially impacting on graft performance. In patients, where the hSV is harvested with pedicle intact, the vasa vasorum is preserved and transmural blood flow restored at graft insertion and completion of CABG. By maintaining blood supply to the hSV wall, apart from oxygen and nutrients, the vasa vasorum may also transport factors potentially beneficial to graft performance. Studies, using either corrosion casts or India ink, have shown the course of vasa vasorum in animal SV as well as in hSV. In addition, there is some evidence that vasa vasorum of hSV terminate in the vessel lumen based on ex vivo perfusion, histological and ultrastructural studies. This review describes the preparation of the hSV as a bypass conduit in CABG and its performance compared with the ITA as well as how and why its patency might be improved by harvesting with minimal trauma in a way that preserves an intact vasa vasorum.     

Patency of the preterm fetal ductus arteriosus is regulated by endothelial nitric oxide synthase and is independent of vasa vasorum in the mouse

Patency of the fetal ductus arteriosus (DA) is maintained in an environment of low relative oxygen tension and a preponderance of vasodilating forces. In addition to prostaglandins, nitric oxide (NO), a potent vasodilator in the pulmonary and systemic vasculatures, has been implicated in regulation of the fetal DA. To further define the contribution of NO to DA patency, the expression and function of NO synthase (NOS) isoforms were examined in the mouse DA on days 17-19 of pregnancy and after birth. Our results show that endothelial NOS (eNOS) is the predominant isoform expressed in the mouse DA and is localized in the DA endothelium by in situ hybridization. Despite rapid constriction of the DA after birth, eNOS expression levels were unchanged throughout the fetal and postnatal period. Pharmacological inhibition of prostaglandin vs. NO synthesis in vivo showed that the preterm fetal DA on day 16 is more sensitive to NOS inhibition than the mature fetal DA on day 19, whereas prostaglandin inhibition results in marked DA constriction on day 19 but minimal effects on the day 16 DA. Combined prostaglandin and NO inhibition caused additional DA constriction on day 16. The contribution of vasa vasorum to DA regulation was also examined. Immunoreactive platelet endothelial cell adhesion molecule and lacZ tagged FLK1 localized to DA endothelial cells but revealed the absence of vasa vasorum within the DA wall. Similarly, there was no evidence of vasa vasorum by vascular casting. These studies indicate that eNOS is the primary source of NO in the mouse DA and that vasomotor tone of the preterm fetal mouse DA is regulated by eNOS-derived NO and is potentiated by prostaglandins. In contrast to other species, mechanisms for DA patency and closure appear to be independent of any contribution of the vasa vasorum.     

Vascular morphogenesis by adult bone marrow progenitor cells in three-dimensional fibrin matrices

The neovascularization of tissues is accomplished by two distinct processes: de novo formation of blood vessels through the assembly of progenitor cells during early prenatal development (vasculogenesis), and expansion of a pre-existing vascular network by endothelial cell sprouting (angiogenesis), the main mechanism of blood vessel growth in postnatal life. Evidence exists that adult bone marrow (BM)-derived progenitor cells can contribute to the formation of new vessels by their incorporation into sites of active angiogenesis. Aim of this study was to investigate the in vitro self-organizing capacity of human BM mononuclear cells (BMMNC) to induce vascular morphogenesis in a three-dimensional (3D) matrix environment in the absence of pre-existing vessels. Whole BMMNC as well as the adherent and non-adherent fractions of BMMNC were embedded in fibrin gels and cultured for 3-4 weeks without additional growth factors. The expression of hematopoietic-, endothelial-, smooth muscle lineage, and stem cell markers was analyzed by immunohistochemistry and confocal laser-scanning microscopy. The culture of unselected BMMNC in 3D fibrin matrices led to the formation of cell clusters expressing the endothelial progenitor cell (EPC) markers CD133, CD34, vascular endothelial growth factor receptor (VEGFR)-2, and c-kit, with stellar shaped spreading of peripheral elongated cells forming tube-like structures with increasing complexity over time. Cluster formation was dependent on the presence of both adherent and non-adherent BMMNC without the requirement of external growth factors. Developed vascular structures expressed the endothelial markers CD34, VEGFR-2, CD31, von Willebrand Factor (vWF), and podocalyxin, showed basement-membrane-lined lumina containing CD45+ cells and were surrounded by alpha-smooth muscle actin (SMA) expressing mural cells. Our data demonstrate that adult human BM progenitor cells can induce a dynamic self organization process to create vascular structures within avascular 3D fibrin matrices suggesting a possible alternative mechanism of adult vascular development without involvement of pre-existing vascular structures.     

Regulation of ornithine decarboxylase and polyamine import by hypoxia in pulmonary artery endothelial cells

In rat lung and cultured lung vascular cells, hypoxia decreases ornithine decarboxylase (ODC) activity and increases polyamine import. In this study, we used rat cultured pulmonary artery endothelial cells to explore the mechanism of hypoxia-induced reduction in ODC activity and determined whether this event was functionally related to the increase in polyamine import. Two strategies known to suppress proteasome-mediated ODC degradation, lactacystin treatment and use of cells expressing a truncated ODC incapable of interacting with the proteasome, prevented the hypoxia-induced decrease in ODC activity. Interestingly, though, cellular abundance of the 24-kDa antizyme, a known physiological accelerator of ODC degradation, was not increased by hypoxia. These observations suggest that an antizyme-independent ODC degradation pathway contributes to hypoxia-induced reductions of ODC activity. When reductions in ODC activity in hypoxia were prevented by the proteasome inhibitor strategies, hypoxia failed to increase polyamine transport. The induction of polyamine transport in hypoxic pulmonary artery endothelial cells thus seems to require decreased ODC activity as an initiating event.     

Angiogenic properties of the chemokine RANTES/CCL5

Atherosclerosis is an inflammatory disease that is one of the leading causes of death in developed countries. This disease is defined by the formation of an atherosclerotic plaque, which is responsible for artery obstruction and affects the heart by causing myocardial infarction. The vascular wall is composed of three cell types and includes a monolayer of endothelial cells and is irrigated by a vasa vasorum. The formation of the vascular network from the vasa vasorum is a process involved in the destabilization of this plaque. Cellular and molecular approaches are studied by in vitro assay of activated endothelial cells and in in vivo models of neovascularization. Chemokines are a large family of small secreted proteins that have been shown to play a critical role in the regulation of angiogenesis during several pathophysiological processes such as ischaemia. Chemokines may exert their regulatory activity on angiogenesis directly by activating the vasa vasorum, or as a consequence of leucocyte infiltration through the endothelium, and/or by the induction of growth factor expression such as that of VEGF (vascular endothelial growth factor). The present review focuses on the angiogenic activity of the chemokines RANTES (regulated upon activation, normal T-cell expressed and secreted)/CCL5 (CC chemokine ligand 5). RANTES/CCL5 is released by many cell types such as platelets or smooth muscle cells. This chemokine interacts with GPCRs (G-protein-coupled receptors) and GAG (glycosaminoglycan) chains bound to HSPGs (heparan sulfate proteoglycans). Many studies have demonstrated, using RANTES/CCL5 mutated on their GAG or GPCR-binding sites, the involvement of these chemokines in angiogenic process. In the present review, we discuss two controversial roles of RANTES/CCL5 in the angiogenic process.     

Macrophage migration inhibitory factor mediates hypoxia-induced pulmonary hypertension

Pulmonary hypertension (PH) is a devastating disease leading to progressive hypoxemia, right ventricular failure, and death. Hypoxia can play a pivotal role in PH etiology, inducing pulmonary vessel constriction and remodeling. These events lead to increased pulmonary vessel wall thickness, elevated vascular resistance and right ventricular hypertrophy. The current study examined the association of the inflammatory cytokine macrophage migration inhibitory factor (MIF) with chronic lung disease and its role in the development of hypoxia-induced PH. We found that plasma MIF in patients with primary PH or PH secondary to interstitial lung disease (ILD) was significantly higher than in the control group (P = 0.004 and 0.007, respectively). MIF involvement with hypoxia-induced fibroblast proliferation was examined in both a human cell-line and primary mouse cells from wild-type (mif ^+/+) and MIF-knockout (mif ^−/−) mice. In vitro, hypoxia-increased MIF mRNA, extracellular MIF protein accumulation and cell proliferation. Inhibition of MIF inflammatory activity reduced hypoxia-induced cell proliferation. However, hypoxia only increased proliferation of mif ^−/− cells when they were supplemented with media from mif ^+/+ cells. This growth increase was suppressed by MIF inhibition. In vivo, chronic exposure of mice to a normobaric atmosphere of 10% oxygen increased lung tissue expression of mRNA encoding MIF and accumulation of MIF in plasma. Inhibition of the MIF inflammatory active site, during hypoxic exposure, significantly reduced pulmonary vascular remodeling, cardiac hypertrophy and right ventricular systolic pressure. The data suggest that MIF plays a critical role in hypoxia-induced PH, and its inhibition may be beneficial in preventing the development and progression of the disease.     

Regulatory effect of AMP-activated protein kinase on pulmonary hypertension induced by chronic hypoxia in rats: in vivo and in vitro studies

Activation of AMP-activated protein kinase (AMPK) plays an important role in cardiovascular protection. It can inhibit arterial smooth muscle cell proliferation and cardiac fibroblast collagen synthesis induced by anoxia. However, the role of AMPK-dependent signalling cascades in the pulmonary vascular system is currently unknown. This study aims to determine the effects of AMPK on pulmonary hypertension and pulmonary vessel remodelling induced by hypoxia in rats using in vivo and in vitro studies. In vivo study: pulmonary hypertension, right ventricular hypertrophy and pulmonary vascular remodelling were found in hypoxic rats. Meanwhile, AMPKα₁ and phosphorylated AMPKα₁ were increased markedly in pulmonary arterioles and lung tissues. Mean pulmonary arterial pressure, index of right ventricular hypertrophy and parameters of pulmonary vascular remodelling, including vessel wall area/total area, density of nuclei in medial smooth muscle cells, and thickness of the medial smooth muscle cell layer were markedly suppressed by AICAR, an AMPK agonist. In vitro study: the expression of AMPKα₁ and phosphorylated AMPKα₁ was increased in pulmonary artery smooth muscle cells (PASMCs) under hypoxic conditions. The effects of PASMC proliferation stimulated by hypoxia were reinforced by treatment with Compound C, an AMPK inhibitor. AICAR inhibited the proliferation of PASMCs stimulated by hypoxia. These findings suggest that AMPK is involved in the formation of hypoxia-induced pulmonary hypertension and pulmonary vessel remodelling. Up-regulating AMPK can contribute to decreasing pulmonary vessel remodelling and pulmonary hypertension induced by hypoxia.     

Imbalances in Mobilization and Activation of Pro-Inflammatory and Vascular Reparative Bone Marrow-Derived Cells in Diabetic Retinopathy

Diabetic retinopathy is a sight-threatening complication of diabetes, affecting 65% of patients after 10 years of the disease. Diabetic metabolic insult leads to chronic low-grade inflammation, retinal endothelial cell loss and inadequate vascular repair. This is partly due to bone marrow (BM) pathology leading to increased activity of BM-derived pro-inflammatory monocytes and impaired function of BM-derived reparative circulating angiogenic cells (CACs). We propose that diabetes has a significant long-term effect on the nature and proportion of BM-derived cells that circulate in the blood, localize to the retina and home back to their BM niche. Using a streptozotocin mouse model of diabetic retinopathy with GFP BM-transplantation, we have demonstrated that BM-derived circulating pro-inflammatory monocytes are increased in diabetes while reparative CACs are trapped in the BM and spleen, with impaired release into circulation. Diabetes also alters activation of splenocytes and BM-derived dendritic cells in response to LPS stimulation. A majority of the BM-derived GFP cells that migrate to the retina express microglial markers, while others express endothelial, pericyte and Müller cell markers. Diabetes significantly increases infiltration of BM-derived microglia in an activated state, while reducing infiltration of BM-derived endothelial progenitor cells in the retina. Further, control CACs injected into the vitreous are very efficient at migrating back to their BM niche, whereas diabetic CACs have lost this ability, indicating that the in vivo homing efficiency of diabetic CACs is dramatically decreased. Moreover, diabetes causes a significant reduction in expression of specific integrins regulating CAC migration. Collectively, these findings indicate that BM pathology in diabetes could play a role in both increased pro-inflammatory state and inadequate vascular repair contributing to diabetic retinopathy.     

Improved pulmonary vascular reactivity and decreased hypertrophic remodeling during nonhypercapnic acidosis in experimental pulmonary hypertension

Pulmonary hypertension (PH) is characterized by pulmonary arteriolar remodeling with excessive pulmonary vascular smooth muscle cell (VSMC) proliferation. This results in decreased responsiveness of pulmonary circulation to vasodilator therapies. We have shown that extracellular acidosis inhibits VSMC proliferation and migration in vitro. Here we tested whether induction of nonhypercapnic acidosis in vivo ameliorates PH and the underlying pulmonary vascular remodeling and dysfunction. Adult male Sprague-Dawley rats were exposed to hypoxia (8.5% O(2)) for 2 wk, or injected subcutaneously with monocrotaline (MCT, 60 mg/kg) to develop PH. Acidosis was induced with NH(4)Cl (1.5%) in the drinking water 5 days prior to and during the 2 wk of hypoxic exposure (prevention protocol), or after MCT injection from day 21 to 28 (reversal protocol). Right ventricular systolic pressure (RVSP) and Fulton's index were measured, and pulmonary arteriolar remodeling was analyzed. Pulmonary and mesenteric artery contraction to phenylephrine (Phe) and high KCl, and relaxation to acetylcholine (ACh) and sodium nitroprusside (SNP) were examined ex vivo. Hypoxic and MCT-treated rats demonstrated increased RVSP, Fulton's index, and pulmonary arteriolar thickening. In pulmonary arteries of hypoxic and MCT rats there was reduced contraction to Phe and KCl and reduced vasodilation to ACh and SNP. Acidosis prevented hypoxia-induced PH, reversed MCT-induced PH, and resulted in reduction in all indexes of PH including RVSP, Fulton's index, and pulmonary arteriolar remodeling. Pulmonary artery contraction to Phe and KCl was preserved or improved, and relaxation to ACh and SNP was enhanced in NH(4)Cl-treated PH animals. Acidosis alone did not affect the hemodynamics or pulmonary vascular function. Phe and KCl contraction and ACh and SNP relaxation were not different in mesenteric arteries of all groups. Thus nonhypercapnic acidosis ameliorates experimental PH, attenuates pulmonary arteriolar thickening, and enhances pulmonary vascular responsiveness to vasoconstrictor and vasodilator stimuli. Together with our finding that acidosis decreases VSMC proliferation, the results are consistent with the possibility that nonhypercapnic acidosis promotes differentiation of pulmonary VSMCs to a more contractile phenotype, which may enhance the effectiveness of vasodilator therapies in PH.     

Endothelial progenitor cells: identity defined?

In the past decade, researchers have gained important insights on the role of bone marrow (BM)-derived cells in adult neovascularization. A subset of BM-derived cells, called endothelial progenitor cells (EPCs), has been of particular interest, as these cells were suggested to home to sites of neovascularization and neoendothelialization and differentiate into endothelial cells (ECs) in situ , a process referred to as postnatal vasculogenesis. Therefore, EPCs were proposed as a potential regenerative tool for treating human vascular disease and a possible target to restrict vessel growth in tumour pathology. However, conflicting results have been reported in the field, and the identification, characterization, and exact role of EPCs in vascular biology is still a subject of much discussion. The focus of this review is on the controversial issues in the field of EPCs which are related to the lack of a unique EPC marker, identification challenges related to the paucity of EPCs in the circulation, and the important phenotypical and functional overlap between EPCs, haematopoietic cells and mature ECs. We also discuss our recent findings on the origin of endothelial outgrowth cells (EOCs), showing that this in vitro defined EC population does not originate from circulating CD133⁺ cells or CD45⁺ haematopoietic cells.     

Impact of coronary vasa vasorum functional structure on coronary vessel wall perfusion distribution

Noncoronary vasa vasorum have been described as networks of microvessels in the wall of arteries and veins. However, we have shown, using microcomputerized tomography (micro-CT) imaging methods, that porcine coronary vasa vasorum have a tree-like branching structure similar to the vasculature in general. In this study, we elucidate functional aspects of coronary vasa vasorum perfusion territories. Three pig hearts were injected with radiopaque Microfil via the coronary sinus to fill the left anterior descending coronary arteries (LADs) retrogradely at atmospheric pressure. In three other hearts, LADs were injected antegradely at 100-mmHg pressure via the left main carotid artery. Additionally, six LADs were injected in vivo with a suspension of 100- or 300-microm-diameter microspheres before harvesting of the hearts and injection of the LADs with Microfil. All harvested LADs were scanned intact with micro-CT (20 microm cubic voxels). The spatial density of vasa vasorum (no. of vasa/mm2) was measured in 20-microm-thick cross sections (at 0.4-mm intervals). Retrogradely injected LADs showed high and uniformly distributed vasa vasorum densities in the adventitia (means +/- SE; 5.38 +/- 0.09 vs. 3.58 +/- 0.1 vasa/mm2 in antegradely prepared LADs; P < 0.001). Antegradely prepared LADs showed patchy distributed, low-vasa-vasorum-density territories especially on the myocardial side of the coronary artery wall (epicardial density: 4.29 +/- 0.13 vasa/mm2 vs. myocardial density: 2.80 +/- 0.1 vasa/mm2, P < 0.001). Microembolization reduced vasa vasorum densities significantly (100-mum-diameter microspheres: 3.26 +/- 0.07 vasa/mm2, P < 0.05; 300-microm-diameter microspheres: 2.66 +/- 0.07 vasa/mm2, P < 0.001 vs. antegrade controls) and increased the size of low-vasa-vasorum-density territories. We conclude that coronary vasa vasorum are functional endarteries not connected via a plexus. This characteristic may have a significant impact on the spatial distribution of perfusion and drainage of the coronary vessel wall.