Endothelial cell-specific knockout of connexin 43 causes hypotension and bradycardia in mice.

Connexin 43 (Cx43) is a protein expressed in a variety of mammalian tissues. However, the lack of specific blockers and the absence of known genetic mutants have hampered the investigation of the function of this protein. Cx43-null mice die shortly after birth, thus preventing functional studies in vivo. Here, we report the generation and characterization of a vascular endothelial cell-specific deletion of the Cx43 gene (VEC Cx43 KO) in mice by using the loxP/Cre system. Using homologous recombination, a mouse line was created carrying loxP sites flanking exon 2 of the Cx43 gene ("floxed" mice). To produce cell specific deletion of the Cx43 gene, these mice were crossed with animals from a line carrying the Tie 2-Cre transgene. The homozygous VEC Cx43 KO mice survived to maturity. However, they were hypotensive and bradycardic when compared with heterozygous VEC Cx43 KO mice, or to the floxed Cx43 gene mice. The hypotension was associated with marked elevation of plasma nitric oxide (NO) levels as well as elevated plasma angiotensin (Ang) I and II. We hypothesize that endothelial cell Cx43 plays a key role in the formation and/or action of NO, and that the elevation of Ang II is a secondary event. The specific cellular basis for the hypotension remains to be established, but our findings support the idea that endothelial Cx43 gap junctions are involved in maintaining normal vascular function; moreover, these animals provide the opportunity to determine more clearly the role of endothelial Cx43 in vascular development and homeostasis.

Microvascular dilation in response to occlusion: a coordinating role for conducted vasomotor responses.

In rat cremasteric microcirculation, mechanical occlusion of one branch of an arteriolar bifurcation causes an increase in flow and vasodilation of the unoccluded daughter branch. This dilation has been attributed to the operation of a shear stress-dependent mechanism in the microcirculation. Instead of or in addition to this, we hypothesized that the dilation observed during occlusion is the result of a conducted signal originating distal to the occlusion. To test this hypothesis, we blocked the ascending spread of conducted vasomotor responses by damaging the smooth muscle and endothelial cells in a 200-microm segment of second- or third-order arterioles. We found that a conduction blockade eliminated or diminished the occlusion-associated increase in flow through the unoccluded branch and abolished or strongly attenuated the vasodilatory response in both vessels at the branch. We also noted that vasodilations induced by ACh (10(-4) M, 0.6 s) spread to, but not beyond, the area of damage. Taken together, these data provide strong evidence that conducted vasomotor responses have an important role in coordinating blood flow in response to an arteriolar occlusion.

A test of the role of flow-dependent dilation in arteriolar responses to occlusion.

At an arteriolar bifurcation, occlusion of one of the branch arterioles has been reported to result in an increase in flow, shear stress, and vasodilation in the opposite unoccluded branch. This dilator response in the unoccluded branch, often referred to as the "parallel occlusion response," has been cited as evidence that flow-dependent dilation is a primary regulator of arteriolar diameter in the microcirculation. It has not been previously noted that, during this maneuver, flow through the feed arteriole would be expected to decrease and logically should cause that vessel to constrict. We tested this prediction in vivo by measuring red blood cell (RBC) velocity and diameter changes in response to arteriolar occlusion in the microcirculatory beds of three preparations: the hamster cheek pouch, the hamster cremaster, and the rat cremaster. In all preparations, a vasodilation was observed in the feed arteriole, despite a decrease in both flow and calculated wall shear stress through this vessel. Unexpectedly, we found that dilation occurred in the unoccluded branch arterioles even in those cases in which RBC velocity and shear stress did not increase in the unoccluded branch arterioles. All values returned to the baseline level after the removal of occlusion. The magnitude of the dilation of the feed and branch arterioles varied between species and tissues, but feed and branch arterioles within a given preparation always responded in a similar way to each other. We conclude from our experiments that mechanisms other than flow-dependent dilation are involved in the vasodilation observed in the microcirculation during occlusion of an arteriolar branch.

Method to create small photo-bleached volumes to monitor blood plasma flow in capillaries.

A method has been developed to examine the movement of plasma in capillaries using intravital microscopy. Spatial transients in fluorescence properties are instantaneously induced by laser photo-bleach pulses after which the convective recovery can be monitored. The plasma is tagged with fluorescent dyes coupled to bovine serum albumin, which is injected well before the measurements and circulates with the blood stream. A laser beam from an argon laser source, set to emit light with a wavelength of 488 nm, is focused on the illumination field diaphragm and creates a spot in the object plane of the microscope. At low laser power, the laser spot is aimed at a blood plasma gap between red blood cells in a capillary segment, using a steerable mirror. Light sensors, coupled to photo-multipliers in the secondary image plane of the microscope, record the light intensity of the moving plasma/dye while the preparation is continuously illuminated with a xenon epi-illuminating set-up. The laser photo-bleach spot is then used to bleach the dye complex within a 5.4 microns segment of the capillary for less than 20 ms. The movement of the bleached plasma bolus is tracked by the photo-sensors, placed sequentially along the capillary. Both dye and red blood cell passage can be detected in the photo-multiplier signals, and the relative velocities of the two blood components can be measured. Measurements reveal that the ratio of transit times between blood plasma and red blood cells is 1.23 (SD = 0.22, N = 18), which is in good agreement with measurements by other techniques.

Determination of capillary tube hematocrit during arteriolar microperfusion.

Intracapillary hematocrit is known to be substantially lower than arterial hematocrit. We hypothesized that capillary hematocrit might be influenced by interactions between plasma macromolecules and the endothelial cell surface. Microvessel perfusion pipettes were inserted in second- or third-order vessels, and capillaries were perfused with three different artificial bloods composed of 50% red cells plus the following suspension media: fetal calf serum (group I), serum albumin plus serum globulins (fractions II and III; group II), and bovine serum albumin plus dextran (group III). The mean hematocrits of the pipette-perfused capillaries averaged close to 50% of the systemic value with all perfusion fluids and were not different from the hematocrits of the capillaries perfused by the animal. These data suggest that bifurcations proximal to the pipette location did not contribute to the reduction in mean tube hematocrit normally seen in the animal. Furthermore, interactions between the plasma macromolecules and the endothelial cell surface do not appear to contribute to the low intracapillary hematocrit. Analysis of the data indicate that the capillary Fåhraeus effect, the network Fåhraeus effect in terminal vessels of the arterial tree, and intracapillary events all contribute to the reduction in intracapillary hematocrit.

A micropipette which allows in situ perfusion of arterioles and capillaries.

In order to investigate capillary physiology, a glass micropipette system was developed that allowed in situ perfusion of microvessels as well as rapid changes of perfusion solutions. Theta tube (WPI, Inc.; 1.5-mm o.d. glass stock capillary tubing which is divided into two hemicylindrical sides by a central glass septum) was pulled to a smaller diameter of approx 300-600 microns and inserted into the shank of a sharpened cannulating micropipette tip constructed from large-bore glass stock (1.6 mm i.d.). The resulting dead volume between the end of the Theta supply tube and the tip of the outer cannulating tip was approximately 90 nl. The perfusate was driven in a circuit from a pressurized feed reservoir down one side of the Theta supply tube pipette and back through the second side into a reservoir maintained at a lower pressure. The pressure gradient between the two reservoirs established a high-volume flow rate and subsequently a short perfusate transit time from the feed to the collection reservoir. The average pressure in the two reservoirs determined the pressure which drove the perfusate from the cannulating tip. At normal pressures and flows, the time required to change perfusion fluid composition at the pipette tip was less than 1 min, and discharge hematocrit of a red blood cell suspension was indistinguishable from the hematocrit measured in the feed reservoir.(ABSTRACT TRUNCATED AT 250 WORDS)

Propagation of vasomotor responses coordinates arteriolar resistances.

We tested the hypothesis that a conduction pathway intrinsic to the arteriolar wall possesses the properties necessary to coordinate vasomotor responses in the microcirculation. Acetylcholine (ACh) or norepinephrine (NE) was iontophoresed onto cheek pouch arterioles (15-35 microns diam) of pentobarbital-anesthetized hamsters, and diameter responses were observed using intravital video microscopy. ACh and NE induced vasodilation and vasoconstriction, respectively, that propagated both upstream and downstream from the site of application. Propagated vasomotor responses decayed with distance along the arterioles; this decay was characterized by mechanical length constants of 1.9 and 1.8 mm for ACh and NE, respectively. Vasodilations and vasoconstrictions initiated on daughter vessels of a branch propagated into parent arterioles that were approximately twice the diameter of the daughter vessels. Iontophoretic stimuli applied simultaneously to paired daughter vessels induced propagated responses that summed linearly in the parent vessel. We conclude that the arteriolar network functions as a highly coordinated syncytium and that diverse vasomotor stimuli can be summed and integrated within the peripheral microvasculature.

Capillary grouping in hamster tibials anterior muscles: flow patterns, and physiological significance.

We have used fluorescein-labeled albumin and epifluorescence videomicroscopy to visualize the organization and flow patterns in the capillaries of a postural muscle, the tibialis anterior, in the pentobarbital-anesthetized hamster. Video tape records were made of overlapping microscope fields comprising portions of the muscle up to two millimeters in length and as much as 180 micron in depth. Three-dimensional reconstructions made from these tapes incorporated observations on flow patterns and geometry of the microvessels. The microcirculation in the tibialis was found to be arranged in repeating modules or 'units', consisting of approximately 15 capillaries supplied by a common arteriole and drained by a common venule. The mean distance from arteriole to venule in a unit was 855 +/- 233 micron (X +/- SD) and the mean capillary length was 805 +/- 330 micron. Flow in the capillaries within a unit was almost entirely concurrent, and there was little interchange of blood flow between the capillaries of adjacent units. The flow in capillaries of adjacent units was both countercurrent and concurrent. For each capillary, a count of the adjacent capillaries with concurrent and countercurrent flow was made. The ratio of concurrent to countercurrent flow varied from 2.4 to 4.3, being greatest at the midpoint of the unit and least at the arterial and venous ends. Our observations suggest that the capillaries of striated muscle are arranged in modules which may function as the fundamental control and distribution elements in the microcirculation.

Inaccuracies in blood flow estimates in microvessels during arteriolar vasoconstriction.

The effect of vasomotor tone on blood flow estimates was evaluated in the hamster cheek pouch and cremaster muscle microcirculation. The products of arteriolar cross-sectional area and red blood cell velocity were calculated in three different cases: (1) at arteriolar bifurcations, (2) in short segments of an arteriole constricted by iontophoretic application of norepinephrine, and (3) at randomly selected second- and third-order arterioles. Vasodilation of the microcirculation was induced by topical application of adenosine. Vasoconstriction was induced by elevation of superfusion solution PO2. If true volume flow is accurately estimated by this method then: the sum of measured branch flows at a bifurcation should equal feed flow; measured flow through constricted arteriolar segments should equal flow proximal or distal to the constricted segment; and, following experimental manipulations, relative changes in estimated flow in second- and third-order arterioles should be equal. Our findings suggest that the blood flow estimates were not always accurate. The sum of branch flows was equal to feed flow only across bifurcations with low or resting vascular smooth muscle tone. During vasoconstriction, feed flow averaged 40% higher than the sum of downstream flows. In addition, estimated flow was 15% lower in constricted segments of an arteriole compared to dilated contiguous segments of the vessel. During alterations in vasomotor state, estimated fractional changes in flow in second- and third-order arterioles differed by more than sixfold. Therefore, blood flow estimates with the dual-slit method may not be reliable under conditions of high vasomotor tone. We speculate that the error may result largely from uncertainties in the diameter measurement.

A computerized system for densitometric analysis of the microcirculation.

An analytical system for measuring size, motion, and light absorption of objects in the field of a microscope is described. The design criteria have been chosen to produce a system that permits acquisition of densitometric information at a number of points within the field and permits computation of important variables from the densitometric data. Relatively simple fiber-optics systems for sampling light intensity at selected locations in the microscope field are described. Computer programs are outlined that permit computation of microvessel diameter, red cell velocity, and hemoglobin oxygen saturation. The sampling systems and programs are quite general in nature and should be applicable to other types of related measurements and computing devices.

Augmented tissue oxygen supply during striated muscle contraction in the hamster. Relative contributions of capillary recruitment, functional dilation, and reduced tissue PO2.

To investigate the relative contributions of alterations in blood flow, capillary density, and tissue PO2 to elevated oxygen delivery in working muscle, we conducted experiments on the suffused hamster cremaster muscle, using in vivo microscopic techniques. Muscle PO2 was measured during striated muscle twitch contraction at 1 Hz. Tissue oxygenation was changed by using suffusion solutions equilibrated with 0%, 5%, 10%, 21%, or 50% oxygen. Contraction caused an increase in capillary density (capillary recruitment), whose magnitude was related to the equilibration gas and, thus, to the suffusate PO2. Capillary recruitment first increased as the oxygen content was raised, peaked with 10% oxygen, and then diminished with higher oxygen content. Arteriolar functional dilation was also observed; when oxygen was raised above 21%, dilation was decreased. The data suggest that oxygen supply is increased primarily by arteriolar conductance changes with low suffusion solution oxygen (0% to 5%), and by capillary recruitment and increased PO2 gradients above 10% oxygen. When vasomotor tone was increased by addition of norepinephrine to the suffusion medium, the changes observed were similar to those observed when oxygen was increased. Therefore, we propose that the altered microvascular responses during vasoconstriction are a function of vascular tone rather than the levels of tissue PO2. A model is proposed which may partially explain the relations among vascular tone, functional dilation, and capillary recruitment. Our data also suggest that tissue PO2 may not be precisely regulated about a narrowly defined set point in this striated muscle but that, instead, tissue PO2 is a dependent variable controlled by the integrated effects of capillary recruitment, functional vasodilation, and altered metabolism.

Tissue PO2 and arteriolar responses to metabolic stimuli during maturation of striated muscle.

Tissue O2 tension (PO2) and small arteriolar diameter were measured in hamsters aged 32, 60, and 80 days. The cremaster muscle was isolated and superfused with a solution equilibrated with 0, 5, or 10% O2 stimulated to contract at 1 Hz. Resting muscle tissue PO2 was proportional to superfusate PO2 and was not different between age groups. The decrease in tissue PO2 during contraction was greatest in adult animals when the superfusate PO2 was low but was equal in all groups when the superfusate PO2 was high. Elevated superfusate PO2 was correlated with a vasoconstriction, the magnitude of which varied inversely with age. Resting and contraction-induced vascular diameter were largest in the youngest animals, relative to maximum diameter, but absolute resting and contraction-induced diameters were similar in all groups. We suggest that tissue PO2 at rest was similar because of an age-associated decrease in fiber O2 consumption to maintain a constant proportionality between O2 supply and demand. The relative stability of tissue PO2 during contraction in young animals might have reflected superior regulation. However, a simple numerical analysis predicts smaller tissue PO2 decreases during contraction in young animals because of short intercapillary distances and other altered O2 supply parameters, even if regulation had been identical in all age groups.

Microvascular adaptations during maturation of striated muscle.

It is well known that capillary density in striated muscle changes during maturation. Capillary density is an important determinant of tissue oxygen supply, the other principal determinants being capillary erythrocyte flow and capillary hematocrit. The microcirculation of the hamster cremaster muscle was studied at different stages of development. We found that the microcirculation of juvenile animals was characterized by small intercapillary distances, short capillary lengths, and tortuous vessels. During maturation, the capillaries elongated and developed the more "typical" parallel pattern. Capillary density decreased from 1,626 +/- 60 capillaries . mm-3 at 35 days of age to 696 +/- 65 capillaries . mm-3 at 132 days; erythrocyte flow per capillary decreased from 1,441 +/- 135 to 583 +/- 47 micrometers 3 . s-1; and capillary hematocrit decreased from 21.5 +/- 0.7 to 14.6 +/- 0.6%. Concomitant with these decreases, the functional reserve increased; in adult muscles, capillary density could increase by 42%, erythrocyte flow per capillary by 457.2%, and capillary hematocrit by 112.4%, compared with 7.7, 20.3, and 24.1%, respectively, in immature animals. These observations show that age significantly modifies microvascular parameters related to tissue oxygen supply and provides an explanation for some conflicting observations in the literature.

Methods for isolation, cannulation, and in vitro study of single microvessels.

A method is described for the isolation and cannulation of microvessels (12-112 micrometers) that permits study, in vitro, of their physiology and pharmacology. Vessels from the hamster cheek pouch, testis, and mesentery and from rat brain have been isolated at 4 degrees C with specially prepared instruments and viewed with an inverted microscope. The vessels were cannulated at one end by equipment developed for renal tubular perfusion. The uncannulated end of the vessel is sealed, and experiments on reactivity and mechanics are carried out at fixed intravascular pressures. The isolated microvessels studied have a modulus of elasticity that is consistent with that observed in large vessels, and they display similar maximal active tension development (approximately 10(6) dyn/cm2). Reactivity to norepinephrine, acetylcholine, and adenosine are in the normal range for microvessels. Spontaneous tone is present, as evidenced by stable tonic contractions as well as phasic contractions in the frequency range of 3-30/min. The vessels display stress activation (myogenic response) consisting of contraction in response to increased intraluminal pressure. Our findings suggest that this preparation will be very useful in elucidating the physiology and pharmacology of the resistance vessels in the terminal vasculature.

Estimates of cellular mechanics in an arterial smooth muscle.

Estimates of force generation or shortening obtained from smooth muscle tissues are valid for individual cells only if each cell is contracting homogeneously and if cells anatomically arranged in series are mechanically coupled. These two assumptions were tested and shown to be valid for the pig carotid media under certain conditions. Homogeneity of cellular responses in carotid strips was estimated from the motion of markers on the tissue during K+ -induced isometric contractions. When tissues were stretched to L0 (the optimum length for force generation), there was little marker movement on stimulation. However, considerable marker movement was observed on stimulation at shorter muscle lengths, reflecting localized shortening or stretching. The mechanical coupling of the very small cells in the media was determined by measuring the dependence of cell length on tissue length. Tissues were fixed with glutaraldehyde during isometric contractions at various tissue lengths (0.4--1.1 x L0). The fixed tissues were macerated with acid and the lengths of the dispersed cells were measured. Cell lengths were broadly distributed at all muscle lengths. However, the direct proportionality between mean cell length and muscle length (as a fraction of L0) indicated that cells which are anatomically in series are coupled force-transmitting structures. We conclude that valid estimates of cellular mechanical function in this preparation can be obtained from tissue measurements at lengths greater than about 0.9L0.