Progression of Arterial Vasa Vasorum from Regulator of Arterial Homeostasis to Promoter of Atherogenesis.

The vasa vasorum (vessels of vessels) are a dynamic microvascular system uniquely distributed to maintain physiological homeostasis of the artery wall by supplying nutrients and oxygen to the outer layers of the artery wall, adventitia, and perivascular adipose tissue, and in large arteries, to the outer portion of the medial layer. Vasa vasorum endothelium and contractile mural cells regulate direct access of bioactive cells and factors present in both the systemic circulation and the arterial perivascular adipose tissue and adventitia to the artery wall. Experimental and human data show that proatherogenic factors and cells gain direct access to the artery wall via the vasa vasorum and may initiate, promote, and destabilize the plaque. Activation and growth of vasa vasorum occur in all blood vessel layers primarily by angiogenesis, producing fragile and permeable new microvessels that may cause plaque hemorrhage and fibrous cap rupture. Ironically, invasive therapies, such as angioplasty and coronary artery bypass grafting, injure the vasa vasorum, leading to treatment failures. The vasa vasorum function both as a master integrator of arterial homeostasis and, once perturbed or injured, as a promotor of atherogenesis. Future studies need to be directed at establishing reliable in vivo and in vitro models to investigate the cellular and molecular regulation of the function and dysfunction of the arterial vasa vasorum.

Recent Developments in Vascular Adventitial Pathobiology: The Dynamic Adventitia as a Complex Regulator of Vascular Disease.

The adventitia, the outer layer of the blood vessel wall, may be the most complex layer of the wall and may be the master regulator of wall physiology and pathobiology. This review proposes a major shift in thinking to apply a functional lens to the adventitia rather than only a structural lens. Human and experimental in vivo and in vitro studies show that the adventitia is a dynamic microenvironment in which adventitial and perivascular adipose tissue cells initiate and regulate important vascular functions in disease, especially intimal hyperplasia and atherosclerosis. Although well away from the blood-wall interface, where much pathology has been identified, the adventitia has a profound influence on the population of intimal and medial endothelial, macrophage, and smooth muscle cell function. Vascular injury and dysfunction of the perivascular adipose tissue promote expansion of the vasa vasorum, activation of fibroblasts, and differentiation of myofibroblasts. This regulates further biologic processes, including fibroblast and myofibroblast migration and proliferation, inflammation, immunity, stem cell activation and regulation, extracellular matrix remodeling, and angiogenesis. A debate exists as to whether the adventitia initiates disease or is just an important participant. We describe a mechanistic model of adventitial function that brings together current knowledge and guides the design of future investigations to test specific hypotheses on adventitial pathobiology.

The microenvironment of the atheroma expresses phenotypes of plaque instability.

Data from histopathology studies of human atherosclerotic tissue specimens and from vascular imaging studies support the concept that the local arterial microenvironment of a stable atheroma promotes destabilizing conditions that result in the transition to an unstable atheroma. Destabilization is characterized by several different plaque phenotypes that cause major clinical events such as acute coronary syndrome and cerebrovascular strokes. There are several rupture-associated phenotypes causing thrombotic vascular occlusion including simple fibrous cap rupture of an atheroma, fibrous cap rupture at site of previous rupture-and-repair of an atheroma, and nodular calcification with rupture. Endothelial erosion without rupture has more recently been shown to be a common phenotype to promote thrombosis as well. Microenvironment features that are linked to these phenotypes of plaque instability are neovascularization arising from the vasa vasorum network leading to necrotic core expansion, intraplaque hemorrhage, and cap rupture; activation of adventitial and perivascular adipose tissue cells leading to secretion of cytokines, growth factors, adipokines in the outer artery wall that destabilize plaque structure; and vascular smooth muscle cell phenotypic switching through transdifferentiation and stem/progenitor cell activation resulting in the promotion of inflammation, calcification, and secretion of extracellular matrix, altering fibrous cap structure, and necrotic core growth. As the technology evolves, studies using noninvasive vascular imaging will be able to investigate the transition of stable to unstable atheromas in real time. A limitation in the field, however, is that reliable and predictable experimental models of spontaneous plaque rupture and/or erosion are not currently available to study the cell and molecular mechanisms that regulate the conversion of the stable atheroma to an unstable plaque.

Education of the clinical embryology laboratory professional: development of a novel program delivered in a laboratory medicine department.

Clinical embryologists are responsible for the handling, evaluation, and care of human gametes and preimplantation embryos within the context of an assisted reproductive technology laboratory. They are integral members of a team of professionals who provide care for fertility patients. Despite the increasing recognition of clinical embryologists as professionals, training requirements, continuing professional development, and appropriate credentialing have lagged in several countries. In many cases, individuals enter the profession with training limited to technical aspects provided by individual laboratory directors through an apprenticeship model. In this article, we present the rationale for rigorous formal training in clinical embryology, introduce CanEMB competencies for practicing professional clinical embryologists that are founded on CanMEDs role principles, and present a nascent Masters of Health Sciences degree program in Laboratory Medicine with a specialization in clinical embryology. This 2-year program has unique features including a Clinical Embryology Skills Development Laboratory, research capstone project, and 200-hour placement within a practicing assisted reproductive technology laboratory. Importantly, this program is delivered through a university-based Department of Laboratory Medicine and Pathobiology in partnership with a Department of Obstetrics and Gynecology. Thus, this program represents a formal acceptance of clinical embryology as a clinical laboratory science. It can be adopted elsewhere to provide a relevant, robust education that will meet current and future needs of the profession.

Fibroblast growth factor-2 promotes in vitro mitral valve interstitial cell repair through transforming growth factor-ß/Smad signaling.

Transforming growth factor (TGF)-ß and fibroblast growth factor (FGF)-2 both promote repair in valve interstitial cell (VIC) injury models; however, the relationship between TGF-ß and FGF-2 in wound repair are not well understood. VIC confluent monolayers were wounded by mechanical injury and incubated separately or in combination with FGF-2, neutralizing antibody to FGF-2, neutralizing antibody to TGF-ß, and betaglycan antibody for 24 hours after wounding. Phosphorylated Smad2/3 (pSmad2/3) was localized at the wound edge (WE) and at the monolayer away from the WE. Down-regulation of pSmad2/3 protein expression via small-interfering RNA transfection was performed. The extent of wound closure was monitored for up to 96 hours. FGF-2 incubation resulted in a significant increase in nuclear pSmad2/3 staining at the WE. Neutralizing antibody to TGF-ß alone or with FGF-2 present resulted in a similar significant decrease in pSmad2/3. Neutralizing antibody to FGF-2 alone or with FGF-2 present showed a similar significant decrease in pSmad2/3; however, significantly more staining was observed than treatment with neutralizing antibody to TGF-ß. Incubation with betaglycan antibody inhibited FGF-2-mediated pSmad2/3 signaling. Wound closure corresponded with pSmad2/3 staining at the WE. Down-regulation of pSmad2/3 via small-interfering RNA transfection significantly reduced the extent to which FGF-2 promoted wound closure. Fibroblast growth factor-2 promotes in vitro VIC wound repair, at least in part, through the TGF-ß/Smad2/3 signaling pathway.

Transforming growth factor-ß regulates the growth of valve interstitial cells in vitro.

Although valve interstitial cell (VIC) growth is an essential feature of injured and diseased valves, the regulation of VIC growth is poorly understood. Transforming growth factor (TGF)-ß promotes VIC proliferation in early-stage wound repair; thus, herein, we tested the hypothesis that TGF-ß regulates VIC proliferation under normal nonwound conditions using low-density porcine VIC monolayers. Cell numbers were counted during a 10-day period, whereas proliferation and apoptosis were quantified by bromodeoxyuridine staining and TUNEL, respectively. The extent of retinoblastoma protein phosphorylation and expression of cyclin D1, CDK 4, and p27 were compared using Western blot analysis. Adhesion was quantified using a trypsin adhesion assay, and morphological change was demonstrated by immunofluorescence localization of α-smooth muscle actin and vinculin. TGF-ß-treated VICs were rhomboid; significantly decreased in number, proliferation, and retinoblastoma protein phosphorylation; and concomitantly had decreased expression of cyclin D1/CDK4 and increased expression of p27. TGF-ß-treated VICs adhered better to substratum and had more vinculin plaques and α-smooth muscle actin stress fibers than did controls. Thus, the regulation of VIC growth by TGF-ß is context dependent. TGF-ß prevents excessive heart valve growth under normal physiological conditions while it promotes cell proliferation in the early stages of repair, when increased VICs are required.

The pathobiology of perivascular adipose tissue (PVAT), the fourth layer of the blood vessel wall.

The perivascular adipose tissue (PVAT) is an adipose tissue depot which surrounds most human blood vessels. It is metabolically active and has both a protective and a pathogenic role in vascular biology and pathobiology. It regulates vascular homeostasis and promotes vascular dysfunction. The purpose of this review is to consider the origin, structure, function, and dysfunction of this unique adipose depot consisting of white (WAT), brown (BAT) and beige adipose tissue, to support the concept that PVAT may be considered the fourth layer of the normal arterial wall (tunica adiposa), in which dysfunction creates a microenvironment that regulates, in part, the initiation and growth of the fibro-inflammatory lipid atherosclerotic plaque. Experimental in-vivo and in-vitro studies and human investigations show that the adipocytes, extracellular matrix, nerve fibers and vasa vasorum found in PVAT form a functional adipose tissue unit adjacent to, but not anatomically separated from, the adventitia. PVAT maintains and regulates the structure and function of the normal arterial wall through autocrine and paracrine mechanisms, that include modulation of medial smooth muscle cell contractility and secretion of anti-inflammatory molecules. PVAT shows regional phenotypic heterogeneity which may be important in its effect on the wall of specific sections of the aorta and its muscular branches during perturbations and various injuries including obesity and diabetes. In atherosclerosis, a pan-vascular microenvironment is created that functionally links the intima-medial atherosclerotic plaque to the adventitia and PVAT beneath the plaque, highlighting the local impact of PVAT on atherogenesis. PVAT adipocytes have inflammatory effects which in response to injury show activation and phenotypic changes, some of which are considered to have direct and indirect effects on the intima and media during the initiation, growth, and development of complicated atherosclerotic plaques. Thus, it is important to maintain the integrity of the full vascular microenvironment so that design of experimental and human studies include investigation of PVAT. The era of discarding PVAT tissue in both experimental and human research and clinical vascular studies should end.

Career transitions: Reflections of former chairs and academic health center leaders.

The 2022 Association of Pathology Chairs Annual Meeting included a live discussion session and a pre-meeting recorded panel webinar sponsored by the Senior Fellows Group (former chairs of academic departments of pathology who have remained active in the Association of Pathology Chairs). The presentation was focused on transition planning for academic health center leaders. Each of the discussion group panelists had served as a pathology department chair as well as in more senior leadership positions, and they provided perspectives based upon their personal experiences. It was noted that such positions are often "at will" appointments of indeterminate length and that those above department chair generally carry greater risks and less stability. Becoming "addicted" to a leadership position was not considered beneficial to the individual or to the institution served and makes transitioning more difficult. Ongoing organizational succession planning was deemed helpful to mitigate such addiction and facilitate personal transition planning. Modes of transitioning discussed included those planned (e.g., voluntary retirement, resignation, administrative advancement) and unplanned (e.g., being "fired"; unexpected personal, health, or family issues). Unplanned transitions were felt to be more difficult, while anticipating when it is time to go and planning for it provided greater personal fulfillment after transition. Many career options were identified after serving in a leadership position, including a return to teaching, research, and/or clinical service; writing; mentoring; becoming more active in professional organizations and boards; philanthropic work; and "reinventing oneself" by moving to another career entirely.

Transforming growth factor-beta regulates in vitro heart valve repair by activated valve interstitial cells.

The regulation of valve interstitial cell (VIC) function in response to tissue injury and valve disease is not well understood. Because transforming growth factor-beta (TGF-beta) has been implicated in tissue repair, we tested the hypothesis that TGF-beta is a regulator of VIC activation and associated cell responses that occur during early repair processes. We used a well-characterized wound model that was created by mechanical denudation of a confluent VIC monolayer to study activation and repair 24 hours after wounding. VIC activation was demonstrated by immunofluorescent localization of alpha-smooth muscle actin (alpha-SMA), and alpha-SMA mRNA levels were quantified by real-time polymerase chain reaction. Proliferation and apoptosis were quantified by bromodeoxyuridine staining and terminal deoxynucleotidyl transferase dUTP nick end labeling, respectively. Repair was quantified by measuring VIC extension into the wound, and TGF-beta expression was shown by immunofluorescent localization of intracellular TGF-beta. Compared with nonwounded monolayers, VICs at the wound edge showed alpha-SMA staining, increased alpha-SMA mRNA content, elongation into the wound with stress fibers, proliferation, and apoptosis. VICs at the wound edge also showed increased TGF-beta and pSmad2/3 staining with co-expression of alpha-SMA. Addition of TGF-beta neutralizing antibody to the wound decreased VIC activation, alpha-SMA mRNA content, proliferation, apoptosis, wound closure rate, and stress fibers. Conversely, exogenous addition of TGF-beta to the wound increased VIC activation, proliferation, wound closure rate, and stress fibers. Thus, wounding activates VICs, and TGF-beta signaling modulates VIC response to injury.

Cardiac fibromas.

Cardiac fibroma is a rare benign nonencapsulated neoplasm of the heart. It occurs mainly in infants and children. It may grow to a large size, which results in a variety of clinical cardiac presentations, including sudden death. Surgical excision prevents recurrence.

Cardiac valve interstitial cells secrete fibronectin and form fibrillar adhesions in response to injury.

BACKGROUND: Fibronectin, an extracellular matrix protein, is associated with the general process of tissue repair and is present in heart valves. In order to understand the cellular mechanisms of heart valve repair, we hypothesized that fibronectin is produced and secreted by valvular interstitial cells (VICs), and when up-regulated in VICs involved in active repair, it is associated with prominent fibrillar adhesions composed of tensin and alpha(5)beta(1) integrin. We investigated the interaction of porcine mitral VICs with the underlying fibronectin matrix and the formation and localization of focal and fibrillar adhesion complexes in an in vitro wound model. METHODS: Confluent monolayers of VICs were wounded with a 1-mm-wide cell scraper, maintained in standard media and 10% fetal bovine serum, and fixed at various time points after wounding. Immunohistochemistry was used to localize fibronectin, paxillin, tensin, and alpha(5)beta(1) integrin. F-actin was localized with an Alexa-Fluor-568-labeled phalloidin. Cells were examined with a scanning confocal laser microscope. RESULTS: In response to in vitro mechanical wounding, migrating VICs at the wound edge expressed cytoplasmic fibronectin compared to nonwounded confluent monolayers. Over 24 to 48 h, fibrils were deposited into the subcellular space. Coincident with this, staining for alpha(5)beta(1) appeared, and tensin redistributed from focal adhesions to fibrillar adhesions, which colocalized with alpha(5)beta(1). CONCLUSIONS: Fibronectin in association with fibrillar adhesions is a component of the matrix that may be secreted by migrating VICs to regulate repair at sites of valve injury.

Undergraduate Specialist Program in Pathobiology at the University of Toronto.

Following a merger of the Departments of Pathology, Clinical Biochemistry, and part of Medical Microbiology, our faculty agreed to deliver a new, unique undergraduate program "Specialist in Pathobiology" at the University of Toronto, in order to teach current concepts of mechanisms of disease to students selected from the large undergraduate science population. The emphasis was on molecular and cellular aspects of pathogenesis and not on the clinical practice of laboratory medicine and pathology. Based on the then new Department of Laboratory Medicine and Pathobiology, we drew upon our large faculty and new recruits in both basic and clinical science to deliver a new curriculum that is unique and dynamic. We began admitting students in 2000, and we have now graduated our 15th class. In this study, we describe our philosophy and goals for the program, and report its success based on student outcomes and innovative course offerings.

Wall tissue remodeling regulates longitudinal tension in arteries.

Changes in blood pressure or flow induce arterial remodeling that normalizes mechanical loads that are imposed on arterial tissue. Arteries are also under substantial longitudinal stretch (axial strain) that may be altered by growth or atrophy of tissues to which they are attached. We therefore tested whether axial strain is also regulated in a negative feedback manner through arterial remodeling. Axial strain in rabbit carotid arteries was increased from 62+/-2% to 97+/-2% without altering other mechanical loads on wall tissues. Strain was reduced within 3 days and completely normalized by 7 days. Remodeling involved tissue elaboration, endothelial cell replication rates were increased by >50-fold and smooth muscle cell replication rates were increased by >15-fold, and substantially elevated DNA, elastin, and collagen contents were recorded. Also, increased rates of apoptosis were indicated by degradation of DNA into oligonucleosomes, and matrix remodeling was reflected in enlarged fenestrae in the internal elastic lamina and increased expression and activation of gelatinases, especially matrix metalloproteinase-2. Intriguingly, reduced axial strain was not normalized, presumably because remodeling processes, apart from cell contraction, are ineffective in decreasing strain, and arterial smooth muscle orientation precludes large effects of contraction on axial strain.

Partial off-loading of longitudinal tension induces arterial tortuosity.

OBJECTIVES: Arterial tortuosity is a frequent manifestation of vascular disease and collateral vessel growth, but its causes are poorly understood. This study was designed to assess the relationship between the development of tortuosity and the mechanical forces that are imposed on arterial tissue. METHODS AND RESULTS: Axial strain in rabbit carotid arteries was reduced from 62+/-2% to 33+/-2% by implanting an interposition graft, prepared from the contralateral carotid, at the downstream end of the artery. Axial strain remained unchanged for 12 weeks; however, all vessels became tortuous because of tissue growth and remodeling. After 7 days, there was a marked elevation in proliferation rates of endothelial and smooth muscle cells; however, increased apoptosis was also detected, and no net accumulation of DNA was observed. Significant accumulations of elastin (24%) and total collagen (26%) occurred by 5 weeks. Gelatin zymography detected upregulation and activation of matrix metalloproteinase-2 (MMP-2), and confocal microscopy revealed enlargement of fenestrae in the internal elastic lamina. MMP inhibition by treatment with doxycycline prevented enlargement of fenestrae and development of tortuosity, and it enabled normalization of axial strain by 5 weeks. CONCLUSIONS: These findings indicate that substantial axial strain is necessary to sustain the morphological stability of arteries, and that a reduction in strain results in arterial tortuosity attributable to aberrant MMP activity.

Heart valve health, disease, replacement, and repair: a 25-year cardiovascular pathology perspective.

The past several decades have witnessed major advances in the understanding of the structure, function, and biology of native valves and the pathobiology and clinical management of valvular heart disease. These improvements have enabled earlier and more precise diagnosis, assessment of the proper timing of surgical and interventional procedures, improved prosthetic and biologic valve replacements and repairs, recognition of postoperative complications and their management, and the introduction of minimally invasive approaches that have enabled definitive and durable treatment for patients who were previously considered inoperable. This review summarizes the current state of our understanding of the mechanisms of heart valve health and disease arrived at through innovative research on the cell and molecular biology of valves, clinical and pathological features of the most frequent intrinsic structural diseases that affect the valves, and the status and pathological considerations in the technological advances in valvular surgery and interventions. The contributions of many cardiovascular pathologists and other scientists, engineers, and clinicians are emphasized, and potentially fruitful areas for research are highlighted.

Conditional cardiac overexpression of endothelin-1 induces inflammation and dilated cardiomyopathy in mice.

BACKGROUND: Myocardial expression of endothelin-1 (ET-1) and its receptors ET(A) and ET(B) is increased in heart failure. However, the role of ET-1 and its signaling pathways in the pathogenesis of myocardial diseases is unclear. METHODS AND RESULTS: Human ET-1 cDNA was placed downstream of a promoter responsive to a doxycycline (DOX)-regulated transcriptional activator (tTA). This line (ET+) was bred with one harboring cardiac myocyte-restricted expression of tTA (alphaMHC-tTA). Myocardial ET-1 peptide levels were significantly increased in binary transgenic (BT, ET+/tTA+) compared with nonbinary transgenic (NBT, ET+/tTA-; ET-/tTA+; ET-/tTA-) or DOX-treated BT littermates (40.1+/-4.7 versus 2.6+/-1.2 fmol/mL, P<0.003). BT mice demonstrated progressive mortality between 5 and 11 weeks after DOX withdrawal, associated with left ventricular dilatation and contractile dysfunction (peak +dP/dT, 4673+/-468 versus 5585+/-658 mm Hg/s, P<0.05). An interstitial inflammatory infiltrate, including macrophages and T lymphocytes, was evident in the myocardium of BT mice, associated with sequential increases in nuclear factor-kappaB translocation and expression of tumor necrosis factor-alpha, interferon-gamma, interleukin-1 and interleukin-6. Significant prolongation of survival was observed with the combined ET(A)/ET(B) antagonist LU420627 (n=8, P<0.05) in BT mice but not the ET(A)-selective antagonist LU135252 (n=5, P=0.9), consistent with an important role for ET(B) in this model. CONCLUSIONS: These are the first data to demonstrate that cardiac overexpression of ET-1 is sufficient to cause increased expression of inflammatory cytokines and an inflammatory cardiomyopathy leading to heart failure and death.

Atherosclerosis and acute coronary syndromes.

In the past 20 years, there has been much new knowledge discovered on the pathogenesis of atherosclerosis and complicated and vulnerable plaques leading to a better understanding of acute coronary syndromes (ACS). The role of thrombosis, lipid metabolism, and inflammation has been investigated at the cellular and molecular levels, resulting in important new diagnostic and therapeutic strategies. The characterization of the role of hemodynamic shear stress and its regulation of cytoskeletal function in endothelial repair and the discovery of endothelial precursor cells (EPCs) derived from the bone marrow have provided new insight into vascular repair. Thus, our knowledge continues to increase, leading to improved prevention, diagnosis, and treatment of ACS.

Nitric oxide promotes in vitro interstitial cell heart valve repair.

BACKGROUND: The cell and molecular biology of heart valve wound repair is not well understood. Valve interstitial cells (IC) are thought to play an important role in valvular wound repair. Because nitric oxide (NO) has been implicated in wound repair, we tested the hypothesis that NO promotes valvular wound repair by examining the presence of the inducible form of nitric oxide synthase (iNOS) in wounded IC monolayers, in vitro. METHODS: Linear denuding wounds were made in confluent monolayers of porcine mitral valve IC plated on glass coverslips. Cultures were fixed at various times (0 to 48 h postwounding), and iNOS was localized in the cells by immunofluorescence microscopy. Cultures were also incubated with iNOS inhibitors L-N(G)-nitroarginine methyl ester (L-NAME) and N-(3-(Aminomethyl)benzyl)acetamidine (1400W), and the extent of wound closure with and without inhibitor was measured at 24, 48 and 72 h postwounding. RESULTS: From 6 to 24 h postwounding, iNOS localization was increased at the wound edge. At 48 h, iNOS was localized beyond the wound edge, into the monolayer, where the intensity of the signal gradually diminished until it was virtually imperceptible. At 24 and 48 h, the inhibition of iNOS with both L-NAME and 1400W resulted in a significant delay in wound closure. CONCLUSION: NO promotes valve wound repair through an effect on IC migration.