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.

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.

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.

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.

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.

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.

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.

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.

Academic Mentorship Builds a Pathology Community.

Since academic mentorship focuses on developing and supporting the next generation of pathologists as well as the existing faculty, it plays a vital role in creating a successful academic pathology department whose faculty deliver quality teaching, research, and clinical care. The central feature is the mentor-mentee relationship which is built on mutual respect, transparency, and a genuine interest from the mentor in the success of the mentee. This relationship is a platform for career development, academic guidance, informed professional choices, and problem solving. Departments of pathology must embrace a culture of effective mentorship so that trainees and faculty members are well mentored. Mentorship should become an academic activity that is valued and rewarded. Departments should create and support formal educational programs that train mentors in mentorship. Effective models of formal mentorship need to be created and evaluated in order to strengthen academic pathology. A successful mentorship culture will provide for a sustainable community of academic pathologists that transmits their best practices to the next generation.

Wnt3a/ß-catenin increases proliferation in heart valve interstitial cells.

BACKGROUND: Valve interstitial cells (VICs), the most prevalent cells in the heart valve, mediate normal valve function and repair in valve injury and disease. The Wnt3a/ß-catenin pathway, important for proliferation and endothelial-to-mesenchymal transition in endocardial cushion formation in valve development, is up-regulated in adult valves with calcific aortic stenosis. Therefore, we tested the hypothesis that Wnt3a/ß-catenin signaling regulates proliferation in adult VICs. METHODS: Porcine VICs were treated with 150 ng/ml of exogenous Wnt3a. To measure proliferation, cells were counted on day 4 posttreatment and stained for bromodeoxyuridine (BrdU) at 24 h posttreatment. ß-Catenin small interfering RNA (siRNA) was used to knock down ß-catenin expression. Apoptosis was measured with terminal deoxynucleotidyl transferase dUTP nick end labeling assay. To assess changes in ß-catenin, cells were stained for ß-catenin at days 1, 3, 6, and 9 posttreatment. Western blot for ß-catenin was performed on whole cell, cytoplasmic, and nuclear extracts at day 4 posttreatment. To measure ß-catenin-mediated transcription, TOPFLASH/FOPFLASH reporter assay was performed at 24 h posttreatment. RESULTS: Wnt3a produced a significant increase in cell number at day 4 posttreatment and in the percentage of BrdU-positive nuclei at 24 h posttreatment. The increase in proliferation was abolished by ß-catenin siRNA. Apoptosis was minimal in all conditions. Wnt3a produced progressively greater ß-catenin staining as treatment length increased from 1 to 9 days. Wnt3a produced a significant increase in ß-catenin protein in both whole cell and nuclear lysates after 4 days of treatment. Wnt3a significantly increased TOPFLASH/FOPFLASH reporter activity after 24 h of treatment. CONCLUSION: Wnt3a/ß-catenin signaling pathway is an important regulator of proliferation in adult VICs.

The progression of calcific aortic valve disease through injury, cell dysfunction, and disruptive biologic and physical force feedback loops.

Calcific aortic valve disease (CAVD) is the most common form of heart valve disease in Western society and results in the second most common cardiovascular surgery performed. Despite its prevalence, high morbidity, and high mortality, the pathogenesis of CAVD still eludes our understanding. This review article brings together experimental in vivo and in vitro as well as human in vivo research in cell and molecular pathobiology to construct an overarching hypothesis regarding the development and progression of CAVD. We focus on injury, cell dysfunction, and disruptive biologic and physical forces, and how they function in positive feedback loops that result in the eventual calcification of the valve. We propose that injury, inflammation, matrix remodeling, and physical forces are all processes that influence each other and alter the normal physiologic functions of a key player in the pathogenesis of CAVD: the valve interstitial cell. We propose that the different phenotypes of the valve interstitial cell play essential roles in the pathogenesis of CAVD. We describe important physiologic processes which become dysfunctional including proliferation, migration, secretion of growth factors, chemokines and cytokines, and matrix remodeling. We also describe the emergence of chondrogenesis and osteogenesis in the fibrotic valve that lead to the severe clinical conditions of CAVD. CAVD appears to have a complex pathogenesis which fortunately can be studied in vitro and in vivo to identify ways to detect, treat, and prevent CAVD.

Fibroblast growth factor-2 promotes in vitro heart valve interstitial cell repair through the Akt1 pathway.

BACKGROUND: Fibroblast growth factor-2 promotes in vitro heart valve interstitial cell repair. Fibroblast growth factor-2 acts through betaglycan which is known to bind both transforming growth factor-ß and fibroblast growth factor-2 at different locations on the molecule. When fibroblast growth factor-2 binds to betaglycan, transforming growth factor-ß binding to betaglycan is reduced, allowing for more transforming growth factor-ß to be available to activate pSmad2/3 which then promotes repair. This study investigates another pathway through which fibroblast growth factor-2 regulates valve interstitial cell repair. METHODS: We used an in vitro model of cell culture disruption. Confluent valve interstitial cell monolayers were disrupted, creating an experimental wound in the confluent monolayer, and incubated in treatments of exogenous fibroblast growth factor-2, anti-fibroblast growth factor receptor antibody, active Akt1, and Akt inhibitor. Valve interstitial cell monolayers were immunohistochemically stained and quantified for nuclear pSmad2/3 at the wound edge. The extent of closure was measured up to 96 h after disruption. RESULTS: Anti-fibroblast growth factor receptor antibody significantly increased both nuclear pSmad2/3 staining at the wound edge and wound closure compared to nontreated control. This increase was less than that seen when valve interstitial cells were treated with fibroblast growth factor-2 and combined treatments of fibroblast growth factor-2 and anti-fibroblast growth factor receptor antibody did not further increase nuclear pSmad2/3 staining compared to fibroblast growth factor-2 alone. This suggested that the regulation of wound closure by fibroblast growth factor-2 also involved pathways other than transforming growth factor-ß/Smad signaling. Treatment with Akt1 significantly increased wound closure, while Akt inhibitor reduced closure as compared to nontreated valve interstitial cells. Fibroblast growth factor-2 and fibroblast growth factor-2 neutralizing antibody up-regulated and down-regulated phosphorylated Akt1 expression in valve interstitial cells, respectively. CONCLUSION: Fibroblast growth factor-2 promotes valve interstitial cell repair in two ways: the fibroblast growth factor-2/fibroblast growth factor-2 receptor interaction through the activation of Akt1 independent of the transforming growth factor-ß/Smad2/3 signaling pathway and the previously described transforming growth factor-ß/Smad signaling.

Cell density regulates in vitro activation of heart valve interstitial cells.

BACKGROUND: Valve interstitial cells, the most prominent cell type in the heart valve, are activated and express α-smooth muscle actin in valve repair and in diseased valves. We hypothesize that cell density, time in culture, and the establishment of cell-cell contacts may be involved in regulating valve interstitial cell activation in vitro. METHODS: To study cell density, valve interstitial cells were plated at passages 3 to 5, at a density of 17,000 cells/22 × 22 mm(2) coverslip, and grown for 1, 2, 4, 7, and 10 days. Valve interstitial cells were stained for α-smooth muscle actin and viewed under confocal microscopy to characterize the intensity of staining. To study time in culture, valve interstitial cells were plated at a 10-fold higher density to achieve similar growth densities over a shorter time period compared with valve interstitial cells plated at low density. α-Smooth muscle actin staining was compared at the same time points between those plated at high and low densities. To confirm valve interstitial cell activation as indicated by α-smooth muscle actin staining, valve interstitial cells were stained for cofilin at days 2, 5, 8, and 14 days postplating. To study the association of transforming growth factor ß with valve interstitial cell activation with respect to cell density, valve interstitial cells were stained for α-smooth muscle actin and transforming growth factor ß at 2, 4, 6, and 8 days postplating. To study the activation of the transforming growth factor ß signaling pathway, valve interstitial cells were stained for pSmad2/3 at days 2, 4, 6, 8, 10, and 12 days postplating. To study cell contacts and activation, subconfluent and confluent cultures of valve interstitial cells were stained for ß-catenin, N-cadherin, and α-smooth muscle actin. Also, whole-cell lysates of subconfluent and confluent valve interstitial cell cultures were probed by Western blot analysis for phospho-ß-catenin at Ser33/37/Thr41, which is the form of ß-catenin targeted for proteosomal degradation. RESULTS: The percentage valve interstitial cells with high-intensity α-smooth muscle actin staining decreases significantly between days 1 and 4, and at confluency, most cells show absent or low-intensity staining, regardless of time in culture. Similar results are obtained with cofilin staining. Transforming growth factor ß and nuclear pSmad2/3 staining in valve interstitial cells decreases concurrently with valve interstitial cell activation as cell density increases. Examining ß-catenin and N-cadherin staining, single valve interstitial cells show no cell-cell contact with strong cytoplasmic staining, with some showing nuclear staining of ß-catenin, while confluent monolayers show strong staining of fully established cell-cell contacts, weak cytoplasmic staining, and absent nuclear staining. The presence of cell-cell contacts is associated with a decreased α-smooth muscle actin. The level of phospho-ß-catenin at Ser33/37/Thr41 is lower in confluent cultures compared with low-density subconfluent valve interstitial cell cultures. CONCLUSION: Cell-cell contacts may inhibit valve interstitial cell activation, while absence of cell-cell contacts may contribute to activation.

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.

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.

The response to valve injury. A paradigm to understand the pathogenesis of heart valve disease.

Human heart valve diseases have become an important topic in cardiovascular pathology and medicine. These diseases have different etiologies and manifestations. However, the most common ones including calcific aortic stenosis have histopathological features that are best characterized as a "response to tissue injury" similar to ones seen in numerous tissues and organs. The valve interstitial cell is the prevalent cell type in the valve and is likely the master cell which ultimately regulates cell and molecular repair processes within the valve that involve autocrine and paracrine processes as well as interactions with the matrix components of the valve. This presentation explores the concept of "response to tissue injury" in understanding the pathogenesis of calcific aortic stenosis.

Common pathogenic features of atherosclerosis and calcific aortic stenosis: role of transforming growth factor-beta.

Calcific aortic stenosis and atherosclerosis have been investigated separately in experimental in vitro and in vivo studies and in clinical studies. The similarities identified in both diseases suggest that similar pathogenic pathways are involved in both conditions. Most current therapeutic studies are focused on statins. The evidence suggests that statin effects on valves may, in large part, be independent of the lipid lowering effects of the drug. There are several molecules that play significant regulatory roles on the development and progression of valve sclerosis and calcification and on growth and complications of atherosclerotic plaques. The purpose of this review is to discuss the pathogenic features of the two conditions, highlight the important similarities, and then review the data that suggest that transforming growth factor-beta may play a key regulatory role in both diseases and that this is worthy of study as a potential therapeutic target for both conditions.

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.