John Kolega

Complex Hemodynamics at the Apex of an Arterial Bifurcation Induces Vascular Remodeling Resembling Cerebral Aneurysm Initiation

Background and Purpose— Arterial bifurcation apices are common sites for cerebral aneurysms, raising the possibility that the unique hemodynamic conditions associated with flow dividers predispose the apical vessel wall to aneurysm formation. This study sought to identify the specific hemodynamic insults that lead to maladaptive vascular remodeling associated with aneurysm development and to identify early remodeling events at the tissue and cellular levels. Methods— We surgically created new branch points in the carotid vasculature of 6 female adult dogs. In vivo angiographic imaging and computational fluid dynamics simulations revealed the detailed hemodynamic microenvironment for each bifurcation, which were then spatially correlated with histologic features showing specific tissue responses. Results— We observed 2 distinct patterns of vessel wall remodeling: (1) hyperplasia that formed an intimal pad at the bifurcation apex and (2) destructive remodeling in the adjacent region of flow acceleration that resembled the initiation of an intracranial aneurysm, characterized by disruption of the internal elastic lamina, loss of medial smooth muscle cells, reduced proliferation of smooth muscle cells, and loss of fibronectin. Conclusions— Strong localization of aneurysm-type remodeling to the region of accelerating flow suggests that a combination of high wall shear stress and a high gradient in wall shear stress represents a “dangerous” hemodynamic condition that predisposes the apical vessel wall to aneurysm formation.

Effects of Direct Current Electric Fields on Cell Migration and Actin Filament Distribution in Bovine Vascular Endothelial Cells

Electric fields exceeding 1 V/cm occur during wound healing, morphogenesis, and tumor growth, and such fields have been shown to induce directional migration of a variety of different cells. However, the mechanism by which electric fields direct cell movement is not yet understood, and the effects on vascular endothelial cells are entirely unknown. We demonstrate that cultured bovine aortic endothelial cells migrate toward the cathode of an applied electric field. Time-lapse microscopic imaging shows that the field suppresses protrusive activity from anode-facing surfaces of the cells while stimulating protrusions from surfaces that face the cathode. The threshold for this response is 1–2 V/cm, similar to field strengths measured in vivo. In addition, fluorescence microscopy shows that lamellipodia projecting toward the cathode are rich in actin filaments. Using quantitative image analysis, we show that the electric field induces a transient 80% increase in the amount of filamentous actin in the cell. Comparison of the distribution of F-actin with total protein distribution indicates that F-actin is asymmetrically distributed in the cytoplasm, being selectively enriched toward the cathode. We propose that physiological electric fields direct cell migration by eliciting an intracellular signal that creates new sites for actin assembly in the cathodal cytoplasm.

Characterization of Critical Hemodynamics Contributing to Aneurysmal Remodeling at the Basilar Terminus in a Rabbit Model

Background and Purpose— Hemodynamic insult by bilateral common carotid artery ligation has been shown to induce aneurysmal remodeling at the basilar terminus in a rabbit model. To characterize critical hemodynamics that initiate this remodeling, we applied a novel hemodynamics–histology comapping technique. Methods— Eight rabbits received bilateral common carotid artery ligation to increase basilar artery flow. Three underwent sham operations. Hemodynamic insult at the basilar terminus was assessed by computational fluid dynamics. Bifurcation tissue was harvested on day 5; histology was comapped with initial postligation hemodynamic fields of wall shear stress (WSS) and WSS gradient. Results— All bifurcations showed internal elastic lamina loss in periapical regions exposed to accelerating flow with high WSS and positive WSS gradient. Internal elastic lamina damage happened 100% of the time at locations where WSS was >122 Pa and WSS gradient was >530 Pa/mm. The degree of destructive remodeling accounting for internal elastic lamina loss, medial thinning, and luminal bulging correlated with the magnitude of the hemodynamic insult. Conclusions— Aneurysmal remodeling initiates when local hemodynamic forces exceed specific limits at the rabbit basilar terminus. A combination of high WSS and positive WSS gradient represents dangerous hemodynamics likely to induce aneurysmal remodeling.

Nascent Aneurysm Formation at the Basilar Terminus Induced by Hemodynamics

Background and Purpose— Hemodynamic insults at arterial bifurcations are hypothesized to play a key role in intracranial aneurysm formation. This study investigates aneurysm-initiating vascular responses at the rabbit basilar terminus subsequent to common carotid artery ligation. Methods— Nine adult female New Zealand white rabbits were subjected to sham, unilateral, or bilateral common carotid artery ligation to produce varying degrees of compensatory basilar artery flow increase. Basilar artery flow velocity and geometry were monitored by transcranial Doppler and rotational angiography, respectively, for 12 weeks after surgery. Bifurcation tissues were harvested at 12 weeks and examined histologically. From the histological sections, we quantified the destructive structural changes at the basilar terminus and correlated them with the basilar artery flow rate increase. Results— Subsequent to common carotid artery ligation, basilar artery flow rate increased by 105% to 900% at the maximum. All common carotid artery-ligated rabbits presented nascent aneurysm formation characterized by a bulge with thinned media and absent internal elastic lamina near the basilar terminus. We defined a nascent aneurysm index based on a multiplicative combination of the local destructive remodeling lengths measured at the nascent aneurysm. The nascent aneurysm index strongly correlated with the increase in basilar artery flow rate with R2=0.91. Conclusion— Without other known predisposition, flow increase alone at the basilar bifurcation can lead to a nascent aneurysm. This nascent aneurysm formation is dose-dependent on basilar artery flow increase.

The Cellular Basis of Epithelial Morphogenesis

Epithelial tissues are ubiquitous in metazoan organisms, performing many different functions and assuming a variety of shapes. This diversity of form and function is ultimately dependent on the behavior of the cells within the epithelia. For example, it is intercellular adhesion and the control of paracellular permeability by cell junctions that permit epithelia to form barriers and act as selective filters. It is cellular polarity that enables absorptive epithelia to extract materials from a particular side of the sheet; it is the collective contributions of cell proliferation, cellular translocation, and changes in cell shape that sculpt epithelia from simple sheets into folds, pouches and tubes. Clearly, a complete understanding of epithelial morphogenesis is inextricably entwined with questions of cell behavior in general, such as how any cell adheres, moves, and maintains its shape. The study of epithelial systems has lent considerable insight into these problems and should continue to do so, just as examination of the behavior and architecture of nonepithelial cells will undoubtedly clarify many aspects of the cellular events underlying epithelial morphogenesis. Although the action of individual cells ultimately shapes epithelial, coordination of that action is necessary for the development of a coherent tissue. Attention must therefore be given to integrative mechanisms in epithelial morphogenesis. How do the many cells in an epithelial sheet act in virtual unison during folding? What defines the boundaries of epithelial invaginations? How does an individual cell detect its position within, and thereby know its role in the morphogenesis of, the epithelial whole of which it is a part? At the most elementary level, epithelial cells interact via their physical attachments to one other. Even such rudimentary communication affects cell shape, movement, and possibly proliferation and also plays a part in the maintenance of epithelial polarity. Additional signals pass among epithelial cells by a number of other mechanisms as well, most notably electrical coupling. However, many questions remain regarding the quality and quantity of what is communicated between epithelial cells. Accordingly, elucidating the means by which supracellular order is maintained in epithelial tissues may still be regarded as the major problem in the study of epithelial morphogenesis.

Molecular alterations associated with aneurysmal remodeling are localized in the high hemodynamic stress region of a created carotid bifurcation.

OBJECTIVEAlthough elevated hemodynamics has been speculated to play a key role in intracranial aneurysm (IA) initiation, little is known about the specific hemodynamic microenvironment that triggers aneurysmal vascular degradation. We previously demonstrated maladaptive remodeling characteristic of IA initiation occurring in hemodynamic regions of combined high wall shear stress (WSS) and high WSS gradient near the apex of an experimentally created carotid bifurcation. This study examines whether this remodeling recapitulates the molecular changes found in IAs and whether molecular changes also correspond to specific hemodynamic environments. METHODSDe novo bifurcations were surgically created using both native common carotid arteries in each of 6 dogs. Bifurcations were imaged 2 weeks or 2 months after surgery by high-resolution 3-dimensional angiography, from which flow fields were obtained by computational fluid dynamics simulations. Subsequently, harvested tissues, demonstrating early aneurysmal changes near the apex, were immunostained for interleukin-1β, endothelial and inducible nitric oxide synthases, nitrotyrosine, and matrix metalloproteinase-2 and -9. Spatial distributions of these molecules were comapped with computational fluid dynamics results. RESULTSThe aneurysmal wall showed decreased endothelial nitric oxide synthase expression compared with surrounding segments, the feeding artery, and native controls, whereas all other markers increased. Anti-CD68 staining indicated the absence of inflammatory cells in the aneurysmal wall. Comapping molecular marker distributions with flow fields revealed confinement of these molecular changes within the hemodynamic region of high WSS and high, positive WSS gradient. CONCLUSIONAneurysm-initiating remodeling induced by combined high WSS and high, positive WSS gradient is associated with molecular changes implicated in IAs.

A model system for mapping vascular responses to complex hemodynamics at arterial bifurcations in vivo.

OBJECTIVECerebral aneurysms are preferentially located at arterial bifurcation apices with complex hemodynamics. To understand disease mechanisms associated with aneurysm initiation, we attempted to establish a causal relationship between local hemodynamics and vascular responses. METHODSArterial bifurcations were surgically created from native common carotid arteries in two dogs, angiographically imaged 2 weeks and 2 months later, and then excised. We characterized local morphological changes in response to specifically manipulated hemodynamics. Computational fluid dynamics simulations were performed on the in vivo images and results mapped onto histological images. RESULTSLocal flow conditions, such as high wall shear stress and high wall shear stress gradient, were found to be associated with vascular changes, including an intimal pad in the flow impingement region and a “groove” bearing the characteristics of an early aneurysm. CONCLUSIONThis novel method of histohemodynamic micromapping reveals a direct correlation between an altered hemodynamic microenvironment and vascular responses consistent with aneurysm development.

Newtonian viscosity model could overestimate wall shear stress in intracranial aneurysm domes and underestimate rupture risk

Objective Computational fluid dynamics (CFD) simulations of intracranial aneurysm hemodynamics usually adopt the simplification of the Newtonian blood rheology model. A study was undertaken to examine whether such a model affects the predicted hemodynamics in realistic intracranial aneurysm geometries. Methods Pulsatile CFD simulations were carried out using the Newtonian viscosity model and two non-Newtonian models (Casson and Herschel-Bulkley) in three typical internal carotid artery saccular aneurysms (A, sidewall, oblong-shaped with a daughter sac; B, sidewall, quasi-spherical; C, near-spherical bifurcation). For each aneurysm model the surface distributions of shear rate, blood viscosity and wall shear stress (WSS) predicted by the three rheology models were compared. Results All three rheology models produced similar intra-aneurysmal flow patterns: aneurysm A had a slowly recirculating secondary vortex near the dome whereas aneurysms B and C contained only a large single vortex. All models predicted similar shear rate, blood viscosity and WSS in parent vessels of all aneurysms and in the sacs of B and C. However, large discrepancies in shear rate, viscosity and WSS among predictions by the various rheology models were found in the dome area of A where the flow was relatively stagnant. Here the Newtonian model predicted higher shear rate and WSS values and lower blood viscosity than the two non-Newtonian models. Conclusions The Newtonian fluid assumption can underestimate viscosity and overestimate shear rate and WSS in regions of stasis or slowly recirculating secondary vortices, typically found at the dome in elongated or complex-shaped saccular aneurysms as well as in aneurysms following endovascular treatment. Because low shear rates and low WSS in such flow conditions indicate a high propensity for thrombus formation and rupture, CFD based on the Newtonian assumption may underestimate the propensity of these events.

Cellular and molecular responses of the basilar terminus to hemodynamics during intracranial aneurysm initiation in a rabbit model.

Background/Aims: Hemodynamics constitute a critical factor in the formation of intracranial aneurysms. However, little is known about how intracranial arteries respond to hemodynamic insult and how that response contributes to aneurysm formation. We examined early cellular responses at rabbit basilar termini exposed to hemodynamic insult that initiates aneurysmal remodeling. Methods: Flow in the basilar artery was increased by bilateral carotid artery ligation. After 2 and 5 days, basilar terminus tissue was examined by immunohistochemistry and quantitative PCR. Results: Within 2 days of flow increase, internal elastic lamina (IEL) was lost in the periapical region of the bifurcation, which experienced high wall shear stress and positive wall shear stress gradient. Overlying endothelium was still largely present in this region. IEL loss was associated with localized apoptosis and elevated expression of matrix metalloproteinases (MMPs) 2 and 9. A small number of inflammatory cells were sporadically scattered in the bifurcation adventitia and were not concentrated in regions of IEL loss and MMP elevation. Elevated MMP expression colocalized with smooth muscle α-actin in the media. Conclusion: The initial vascular response to aneurysm-initiating hemodynamic insult includes localized matrix degradation and cell apoptosis. Such destructive remodeling arises from intrinsic mural cells, rather than through inflammatory cell infiltration.

Progressive aneurysm development following hemodynamic insult

OBJECT Hemodynamic insult has been speculated to be a key factor in intracranial aneurysm formation; however, it is unclear whether a sustained insult is necessary. The authors examined whether aneurysmal degradation would continue despite the normalization of wall shear stress (WSS) by adaptive outward vascular remodeling. METHODS Twenty-five rabbits underwent either sham operation (5 animals) or bilateral common carotid artery ligation (20 animals) to augment basilar artery (BA) flow. Basilar termini (BTs) were harvested at 5 days and 3, 12, and 27 weeks postoperation. Histological changes at the BTs were quantified using an aneurysm development score (ADS) wherein the luminal length of the vessel wall exhibiting internal elastic lamina (IEL) loss, media thinning (> 30% media loss), and bulging was multiplied by the percentage of media thinning divided by the BA diameter. This score and its component variables were evaluated over the specified time points and compared with the WSS time course obtained from multiple angiography and BA flow velocity measurements. RESULTS Serial examination of histological sections from the ligation group (17 rabbits survived the procedure) demonstrated localized, progressive, degenerative, and aneurysmal changes at the BTs. Prominent IEL loss was observed in BT specimens from all ligated animals. Media thinning and luminal bulging significantly progressed over the 27-week follow-up. The composite ADS significantly increased over the study period, indicating progressive aneurysm development, although the WSS returned to preligation baseline values within 5 weeks of ligation. CONCLUSIONS Hemodynamic insult can elicit a pathological vascular response leading to a self-sustaining aneurysmal remodeling that does not require persistence of the original inciting factor to continue its pathological progression.