Skin Microvasculature Research Articles

Potentiation of Photodynamic Therapy Antitumor Activity in Mice by Nitric Oxide Synthase Inhibition Is Fluence Rate Dependent

The effects of systemic administration of the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine (L-NNA) in combination with photodynamic therapy (PDT) on tumor response, tumor oxygenation and tumor and normal skin perfusion were studied in C3H mice bearing subcutaneous radiation-induced fibrosarcoma tumors. Photodynamic therapy was carried out using the photosensitizer Photofrin (5 mg/kg) in conjunction with a low fluence rate (30 mW/cm2) and a high fluence rate (150 mW/cm2) protocol at a total fluence of 100 J/cm2. Low fluence rate PDT produced approximately 15% tumor cures, a response not significantly altered by administration of 20 mg/kg L-NNA either 5 min before or after PDT. In contrast, high fluence rate PDT produced no tumor cures by itself, but addition of L-NNA either pre- or post-PDT resulted in approximately 30% and approximately 10% tumor cures, respectively. The L-NNA by itself tended to decrease tumor pO2 levels and perfusion, but statistically significant differences were reached only at one time point (1 h) with one of the oxygenation parameters measured (% values < 2 mm Hg). Photodynamic therapy by itself decreased tumor oxygenation and perfusion more significantly. Addition of L-NNA before PDT further potentiated this effect. The L-NNA exerted its most striking effects on the PDT response of the normal skin microvasculature. Low fluence rate PDT caused severe and lasting shut-down of skin microvascular perfusion. With high fluence rate PDT, skin perfusion was initially decreased but recovered to persistent normal levels within 1 h of treatment. Administration of L-NNA reversed this response, converting it to complete and lasting vascular shut-down identical to that achieved with low fluence rate PDT. This effect was somewhat L-NNA dose dependent but was still marked at a dose of 1 mg/kg. It occurred whether L-NNA was given before or after PDT. The L-NNA did not alter the long-term vascular response of skin to low fluence rate PDT. The ability of L-NNA to correspondingly improve tumor response and severely limit skin vascular perfusion following high fluence rate PDT, while providing no benefit for the low fluence rate protocol, suggests that vascular changes in the tumor surrounding normal tissue contribute to the enhanced tumor curability with adjuvant L-NNA treatment.

CGRP and nitric oxide of neuronal origin and their involvement in neurogenic vasodilatation in rat skin microvasculature.

1. Sensory nerves are important for the initiation of neurogenic inflammation and tissue repair. Both calcitonin gene-related peptide (CGRP) and nitric oxide (NO) have been implicated in neurogenic vasodilatation and inflammatory responses. 2. A blister model in the rat hind footpad was used as a site to induce neurogenic vasodilatation in response to antidromic electrical stimulation of the sciatic nerve. Blood flux was monitored with a laser Doppler flow monitor. 3. The quantitative contributions of CGRP and NO to vasodilatation were examined by use of the CGRP receptor antagonist CGRP8-37 and NO synthase inhibitors 7-nitroindazole (7-NI), 3-bromo 7-NI and N(G)-nitro L-arginine methyl ester (L-NAME). The potential modulatory role of endothelin was examined by use of the ET(A) receptor antagonist BQ-123. 4. CGRP8-37 (10 microM) was perfused over the blister base before nerve stimulation and continuously throughout the post-stimulation period, resulting in a significant reduction (41%) in the blood flux vascular response. 5. Pretreatment with the specific neuronal NO synthase inhibitors, 7-NI and 3-bromo 7-NI (10 mg kg(-1), i.v.), and of the non-specific L-NAME (100 microM), resulted in significant inhibition of the blood flux response (36%, 72% and 57% decrease, respectively). In contrast, 7-NI treatment in young rats pretreated with capsaicin had no further effect on the vascular response, suggesting that the source of NO is the sensory nerves. 6. BQ-123 (10 microM) significantly enhanced the stimulation-induced blood flux response (61% increase). When 7-NI was co-administered with either CGRP8-37 or BQ-123, the drug actions were additive, suggesting that there was no interaction between NO and CGRP or endothelin. 7. These data suggest that both NO and CGRP participate in neurogenic vasodilatation in rat skin microvasculature and that this response is modulated by endogenous endothelin.

Attenuation of neurogenic vasoconstriction by nitric oxide in hindlimb microvascular beds of the rat in vivo.

There is evidence that sympathetic nerve activity leads to endothelium-derived nitric oxide release, which in turn attenuates neurogenic vasoconstriction. Here we tested in vivo (1) whether the magnitude of the vasoconstriction induced by N(G)-nitro-L-arginine methyl ester given systemically is altered when ongoing sympathetic activity is abolished by sectioning the lumbar sympathetic trunk, and (2) whether hindlimb sympathetic vasoconstriction elicited by electrical stimulation of the lumbar sympathetic trunk is enhanced after inhibition of nitric oxide synthesis. Blood flow in the microvascular beds of hairless skin and skeletal muscle of the rat hindlimb was measured with laser Doppler flowmetry. Sectioning the lumbar sympathetic trunk resulted in an increase of blood flow in both tissues, indicating that tonic neurogenic vasoconstriction was abolished. Inhibition of nitric oxide synthesis resulted in vasoconstriction in both vascular beds. This vasoconstriction was more pronounced after abolition of sympathetic activity than with intact sympathetic supply in skin but was smaller in skeletal muscle. The vasoconstriction elicited by graded electrical stimulation of the centrally sectioned lumbar sympathetic trunk with frequencies less than 5 Hz was significantly enhanced after blockade of nitric oxide in skeletal muscle but not in skin microvasculature. These findings suggest that under physiological conditions, sympathetic nerve impulses directly promote the release of nitric oxide in skeletal muscle but not in cutaneous blood vessels. Therefore, basal nitric oxide release is probably in part dependent on sympathetic activity in skeletal muscle, whereas it appears to be mainly due to flow-dependent shear stress in hairless skin microvasculature.

Thrombin Generation Measured Ex Vivo Following Microvasculor Injury Is Increased in SLE Patients with Antiphospholipid-protein Antibodies

Antiphospholipid-protein antibodies (APA) include lupus-type anticoagulant (LA) and antibodies recognizing complexes of anionic phospholipids (e.g. cardiolipin) and proteins (e.g. prothrombin and beta2-glycoprotein I). The presence of APA is associated with an increased risk of both arterial and venous thrombosis. However, the pathogenic mechanism leading to thrombosis in patients with APA remains unclear. We studied 32 patients with systemic lupus erythematosus (SLE) who were divided into two groups depending on the presence (n = 19) or absence (n = 13) of APA. Healthy volunteers (n = 12) matched by age and sex served as controls. In all subjects LA and IgG class anticardiolipin antibodies (ACA) were determined. Thrombin generation was monitored ex vivo measuring fibrinopeptide A (FPA) and prothrombin fragment F1 + 2 (F1 + 2) in blood emerging from a skin microvasculature injury, collected at 30 second intervals. In subjects with antiphospholipid antibodies mean FPA and F1 + 2 concentrations were significantly higher at most blood sampling times than in controls. In some SLE patients with APA the process of thrombin generation was clearly disturbed and very high concentrations of fibrinopeptide A were detected already in the first samples collected. Two minutes after skin incision SLE patients without APA produced slightly more FPA, but not F1 + 2, as compared to healthy subjects. Mathematical model applied to analyze the thrombin generation kinetics revealed that APA patients generated significantly greater amounts of thrombin than healthy controls (p = 0.02 for either marker). In contrast, in the same patients generation of thrombin in recalcified plasma in vitro was delayed pointing to the role of endothelium in the phenomenon studied. In summary, these data show for the first time that in SLE patients with antiphospholipid-protein antibodies thrombin generation after small blood vessel injury is markedly increased. Enhanced thrombin generation might explain thrombotic tendency observed in these patients.

Mechanisms underlying the vascular activity of β-amyloid protein fragment (βA425–35) at the level of skin microvasculature

Mechanisms underlying the vascular activity of β-amyloid protein fragment (βA425–35) at the level of skin microvasculature

Mechanisms underlying the vascular activity of β-amyloid protein fragment (βA 425–35) at the level of skin microvasculature

Deposition of β-amyloid protein ( βA 4) in extracellular senile plaques is a pathologic hallmark of Alzheimer's disease (AD). The neurotoxic effect of / gbA 4 has been ascribed to a discrete 11-amino acid internal sequence ( βA 425–35). Substance P (SP) has have been to be depleted in the brain of AD patients while its presence was found to protect against the neurodegenerative effect of βA 425–35. Our previous studies, in vivo, in aged rats showed that βA 425–35 exhibits a potent vasoconstrictor (VC) effect in rat skin microvasculature and can prevent SP but not calcitonin gene-related peptide (CGRP) from inducing a vasodilator (VD) response. It was postulated that βA 425–35 might be interacting with SP at the level of the second messenger system via the phosphoinositide pathway. Using a blister model of inflammation in the rat hind footpad, we examined the ability of βA 425–35 to modulate the vascular activity of bradykinin (BK) and serotonin (5-HT) which also activate the phosphoinositide pathway. In addition, the role of nitric oxide (NO), endothelin (ET, an endothelium-derived constrictor factor) and protein kinase C (PKC) in the vascular effects of βA 425–35 were examined using the NO synthase inhibitor, N G-nitro- l-arginine ( l-NOARG), the ET-receptor antagonist, BQ-123, and the PKC inhibitor, bisindolylmaleimide (BIM) respectively. Changes in microvascular blood flow were monitored using laser Doppler flowmetry and the area within the response curve measured. The results showed that βA 425–35 (10 μM) induced a VC effect and inhibited the subsequent VD response to BK (10 μM) and 5-HT (1 μM) in a similar fashion to its effect on SP (1 μM). In the presence of l-NOARG (100 μM), the VD effect of SP was reduced and further attenuated after perfusion of βA 425–35. Superfusion of the blister base with BQ-123 (10 μM) or BIM (1 μM) prior to and during perfusion with βA 425–35 abolished its VC effect and allowed SP to induce a normal VD response in both young and old rats. Based on these results, we suggest that the vascular activity of the active fragment, / gbA 425–35, is mediated by ET via activation of PKC. This study provides new findings which may help to elucidate the signal transduction mechanisms involved in the vascular activity of βA 425–35. The relevance of these mechanisms to those underlying the pathological effects of / gbA 4 and their significance in AD remains to be determined.

Inhibition of Thrombin Generation by Aspirin Is Blunted in Hypercholesterolemia

Recent evidence indicates that aspirin inhibits thrombin generation in clotting blood. We noticed that this effect was less pronounced in patients with hypercholesterolemia. The aim of the study was to prove this observation. The effects of aspirin on thrombin generation were evaluated in (1) 46 healthy volunteers, 2 hours after ingestion of a single, 500-mg dose and (2) 28 survivors of myocardial infarction who took 300 mg aspirin/d for 2 weeks. In both populations, two well-matched subgroups were distinguished, using a serum cholesterol level of 6.2 mmol/L (240 mg/dL) and an LDL cholesterol level of 4.0 mmol/L (155 mg/dL) as borderline. Thrombin generation was monitored ex vivo in blood emerging from a skin microvasculature injury and additionally, in a single-dose study in vitro in recalcified plasma. Aspirin depressed thrombin generation in the group of subjects with serum cholesterol < 6.2 mmol/L and LDL cholesterol < 4.0 mmol/L but not in the group with high blood cholesterol levels. Inhibitory effects of aspirin were more pronounced after the 2-week treatment than after a single dose. There was a significant correlation between total serum cholesterol or LDL cholesterol and total amount of thrombin generated after aspirin treatment. In subjects with high blood cholesterol levels, thrombin generation was not affected by aspirin. Blunting of aspirin action in hypercholesterolemia might be explained by (1) alterations in platelet lipid-protein matrix that render their membrane proteins less accessible for acetylation by aspirin and (2) changes in composition and structure of plasma lipoproteins that diminish the chance of aspirin to interact with prothrombin.

Effect of chronic sciatic nerve lesion on the neurogenic inflammatory response in intact and acutely injured denervated rat skin.

A supersensitivity to the neuropeptide substance P (SP) has been shown to develop in post-terminal membranes of many denervated tissues. This study examined changes in the sensitivity of post-terminal vascular receptors to SP and calcitonin gene-related peptide (CGRP) in rat skin microvasculature following sciatic nerve section. In anaesthetised rats, 0.5 cm of sciatic nerve in the right mid-thigh region was removed. Two weeks later, SP (100 microM) and sodium nitroprusside (SNP, 1 mM), a direct smooth muscle vasodilator, were introduced into denervated intact footpad skin, via the electrophoresis technique. Laser doppler flowmeter was used to record changes in relative blood flow in the rat hind footpad. The results showed a significant increase in SP response over controls and slight increase in smooth muscle reactivity as determined by an increase in the vascular response to SNP. In another set of experiments, the sensitivity of post-terminal receptors was examined over a 4 weeks period in an acutely injured footpad skin of sciatic nerve lesioned rats. A vacuum-induced blister was raised on the hind footpad and SP, CGRP (each at 1 microM) or SNP (100 microM) were superfused over the blister base. In nerve lesioned rats, using the acutely injured footpad skin model, the results showed a reduction in the vascular responses to SP, CGRP and SNP. The response to SP continued to decrease over time reaching 22% of control values by 4 weeks. Responses to SNP and CGRP were reduced to 53% and 45% respectively by 2 weeks and then improved to 75% of control values by 4 weeks. Possible contributions of sympathetic efferents and the saphenous nerve to these reduced responses in acutely injured skin of nerve lesioned rats were examined using guanethidine (50 mg/kg i.p.) or sectioned saphenous nerve respectively. These procedures did not significantly modify the reduced vascular responses in the blister base of lesioned rats. Possible activation of endogenous opioids and/or the release of endothelin due to blister induction in nerve lesioned rats was examined using naloxone and the endothelin receptor antagonist, BQ-123, respectively. Treatment with naloxone increased SP response in lesioned rats to 41% of control value with no change in smooth muscle reactivity. BQ-123 significantly increased the responses to SP and SNP to 51% and 100% of their own control values respectively. It is concluded that supersensitivity of post-terminal vascular receptors develops in intact skin following chronic nerve lesion. On the other hand, acute injury of the denervated skin area induces activation of endogenous inhibitory modulatory mechanisms that masks this supersensitivity.

Role of nitric oxide in the actions of substance P and other mediators of inflammation in rat skin microvasculature

The role of nitric oxide in inflammatory responses to substance P and other mediators of inflammation was examined in rat skin microvasculature in a blister base raised on the hind footpad. Superfusion of substance P (1 μM) over the blister base caused an increase in plasma extravasation and a vasodilator response which was not maintained. N G-Nitro- l-arginine (100 μM), an inhibitor of nitric oxide biosynthesis, attenuated vasodilatation and plasma extravasation due to substance P. The inactive N G-nitro- d-arginine was without effect. Neurokinin A (1 μM), 5-hydroxytryptamine (1 μM), ATP (50 μM) and vasoactive intestinal polypeptide (1 μM) elicited vasodilatation, which for vasoactive intestinal polypeptide was maintained even after washout. 5-Hydroxytryptamine and neurokinin A, but not ATP or vasoactive intestinal polypeptide, significantly increased plasma extravasation. Vasodilatation to neurokinin A, 5-hydroxytryptamine and ATP, and the increase in plasma extravasation due to neurokinin A and 5-hydroxytryptamine were unaffected by N G-nitro- l-arginine (100 μM), whereas vasodilatation due to vasoactive intestinal polypeptide was significantly attenuated. These findings suggest that in rat skin microvasculature in vivo, nitric oxide is involved in vasodilator responses due to substance P and vasoactive intestinal polypeptide, and plasma extravasation due to substance P, but does not contribute significantly to vasodilatation induced by neurokinin A, 5-hydroxytryptamine or ATP, or to plasma extravasation induced by neurokinin A or 5-hydroxytryptamine.

The effect of aging on skin blood flow in the Wistar-Kyoto rat.

Using laser Doppler techniques in man, we have previously demonstrated that skin blood flow decreases linearly with age. In previous work, we have shown similarities in skin blood flow properties between man and the rat. Our goal was to assess a possible aging effect of skin blood flow in the rat. We determined skin blood flow sequentially in 13 Wistar-Kyoto rats, beginning at 3 months of age, then at 6, 12, 15, and 18 months. We measured flow on the back, the base of the tail, the upper leg, all nutritively (NUTR) perfused sites, and on the plantar paw surface, which is perfused chiefly by arteriovenous anastomotic (AVA) capillaries. We measured flow, microvascular volume and red blood cell velocity at basal temperature and also heated the skin locally to 44 degrees C, to elicit maximal vasodilation. Skin blood flow declined in a linear fashion with increasing age. The decrease was in the order of 15% over 18 months and occurred at both NUTR sites and the paw. The decrease was due to reduced microvascular volume, presumably representing loss of skin capillaries. Red blood cell velocity was not reduced; in fact, it was significantly increased at several NUTR sites. The WKY rat provides an excellent model of aging of the skin microvasculature. The changes we have demonstrated, while not identical to those we have previously demonstrated in man, are sufficiently similar to permit further exploration of the mechanisms of these changes.

βA 425–35 modulates substance P effect on rat skin microvasculature in aged rats: pharmacological manipulation using SEC-receptor ligands

The primary constituent of the senile plaque core in Alzheimer's disease (AD) is the β-amyloid protein (βA 4). A discrete 11 amino acid fragment of the βA 4, βA 425–35, has been implicated in mediating in vitro neurotoxicity and an inflammatory response surrounding senile plaques in AD via interaction with the Serpin Enzyme Complex (SEC) receptor. Substance P (SP), a neuropeptide of the tachykinin family and a major mediator of neurogenic inflammation, shows sequence homology to βA 425–35 and has been shown to protect against the neurotoxicity of β-amyloid. SP also competes with βA 425–35 for binding to the SEC-receptor. SP neurons have also been found to be depleted in AD. Using a blister model of inflammation in the rat hind footpad, we have examined the effect of βA 425–35 and its interaction with SP in rat skin microvasculature and determined age-related changes to these phenomena. In addition, pharmacological manipulation of these responses using SEC-receptor ligands (peptide 105Y and 105C) was also undertaken. Because of the evidence for co-existence and co-release of SP and calcitonin gene-related peptide (CGRP) from the peripheral terminals of sensory nerves, it was of interest to examine the interaction of CGRP with βA 425–35 on rat skin microvasculature. βA 425–35 (10 μM) was perfused over the base of a blister raised on the hind footpad of anaesthetised young and old rats. This was followed by perfusion of SP (1 μM) or CGRP (1 μM) after Ringer's solution. Relative blood flow was monitored using a Laser-Doppler Flowmeter. Peptide 105Y or Peptide 105C (100 μM) was perfused over the blister base prior to perfusion of βA 425–35 and SP. The results showed that βA 425–35 induced a vasoconstrictor effect on rat skin microvasculature that was significantly greater in old than in young rats. Furthermore, βA 425–35 prevented a subsequent SP-, but not CGRP-induced vasodilator response in old rats. Perfusion of SEC receptor ligands (peptide 105Y and 105C) or brief perfusion of SP prior to βA 425–35 resulted in a reduction in the vasoconstrictor effect of βA 425–35 and allowed a subsequent SP perfusion to induce a small vasodilator response. This pharmacological manipulation was most evident using peptide 105C. This study demonstrates that skin can provide an index of the effect of βA 425–35 on the microvasculature in vivo and suggests that βA 425–35 prevents SP from inducing a vasodilator effect in old rats through an interaction with the SEC receptor. These modulatory effects can be pharmacologically manipulated using the SEC receptor ligands peptide 105Y, 105C and SP. This blister model of inflammation should be useful for examining further the nature of these interactions and their significance in AD.

Effect of BQ-123 and Ro 47-0203 (bosentan) on endothelin-induced vasoconstriction in the rat skin

The effectiveness of intradermal (i.d.) BQ-123 (cyclo[ d-Asp-Pro- d-Val-Leu- d-Trp]) and i.d. Ro 47-0203 (bosentan, 4- tert-butyl- N-[6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-2,2′-bipyrimidin-4-yl]-benzene-sulfonamide) has been evaluated on local microvascular responses to endothelin-1 and endothelin-3, measured by a multiple site 133Xe clearance technique in rat skin in vivo. Intradermal injection of endothelin-1 (0.3 pmol/site) and endothelin-3 (10 pmol/site) induced a similar (approximately 50–60%) decrease in basal blood flow in rat skin. BQ-123 (3–1000 pmol/site), a selective endothelin ET A receptor antagonist, caused a significant dose-dependent decrease in the vasoconstriction induced by endothelin-1 ( P < 0.05) but was less effective on vasoconstriction induced by endothelin-3. Bosentan (3–100 pmol/site), a new non-peptide mixed antagonist of endothelin ET A and endothelin ET B receptors, significantly reduced the vasoconstriction induced by endothelin-1 but was less effective than BQ-123. BQ-123 and bosentan were similarly effective as antagonists of endothelin-3. BQ-123 and bosentan had no effect on basal blood flow and no inhibitory activity on vasoconstriction induced by vasopressin (0.03 pmol/site) or phenylephrine (300 pmol/site). These findings indicate that BQ-123 and bosentan are effective and selective inhibitors of the vasoconstriction induced by endothelins in the rat skin microvasculature.

Nitric oxide and human skin blood flow responses to acetylcholine and ultraviolet light

Nitric oxide (NO) is a potent endogenous vasodilator of large blood vessels but its role in the physiological and pathological control of the human microcirculation is not known. This study was designed to assess whether NO contributes to the control of blood flow in the human skin microcirculation in vivo. Local changes in blood flow were measured in the forearm skin microcirculation of normal volunteers. The responses to agents injected intradermally were assessed with a laser Doppler flow probe. NO was generated in local areas of skin by the injection of the NO donor, sodium nitroprusside. Endogenous NO was generated by the injection of acetylcholine and the exposure of skin to ultraviolet light (UVB) to stimulate the constitutive and inducible forms of nitric oxide synthase (NOS), respectively. The skin microvasculature was comparatively insensitive to exogenous NO derived from the NO donor sodium nitroprusside, being 10,000-fold more sensitive to the vasodilator prostaglandin PGE2 (P < 0.001). However, the rapid onset, dose-dependent local vasodilation caused by acetylcholine was blocked by the NOS inhibitor NG-monomethyl-L-arginine, L-NMMA (P < 0.05), but not by the cyclooxygenase inhibitor indomethacin. The delayed local blood flow response to UVB was attenuated by either L-NMMA or indomethacin (P < 0.05 in each case). The UVB response was abolished by a combination of L-NMMA and indomethacin or by local, topical corticosteroid treatment (P < 0.01 in each case). This study indicates that NO increases blood flow in the human microcirculation in vivo. NOS inhibition attenuated both the rapid blood flow response to acetylcholine and the delayed response to UVB. Both NOS and cyclooxygenase contributed to the erythema response to UVB, and dual inhibition of both enzymes would explain the mechanism of action of the corticosteroid in this model of inflammation.

Antiplatelet drugs and generation of thrombin in clotting blood

Platelets participate in formation of thrombin through secretion of coagulation factors and by providing a catalytic surface on which prothrombinase complex is assembled. We studied the effects of four antiplatelet drugs on thrombin formation in healthy volunteers. Thrombin generation was monitored both in vitro--in recalcified plasma--and ex vivo--in blood emerging from a standardized skin microvasculature injury, which also served to determine bleeding time. A mathematical model has been developed to describe the latter reaction. It is based on estimation of the rate of increase in fibrinopeptide A (FPA), a specific marker of thrombin activity, in blood emerging from skin incisions. Two hours after the ingestion of 500 mg of aspirin, thrombin formation became significantly impaired both in vitro and ex vivo. In contrast, 2 hours after the oral administration of placebo, indomethacin 50 mg, or OKY-046 (a thromboxane synthase inhibitor) 400 mg, thrombinogenesis remained unaltered. Ticlopidine, studied either 3 hours after 500 mg oral administration, or after 5 days of intake at a daily dose of 500 mg, had no effect on thrombin generation. Thus, aspirin, contrary to other antiplatelet drugs, depresses thrombin formation in clotting blood, a phenomenon that might be of clinical relevance. It is suggested that aspirin exerts this effect by acetylating prothrombin and/or macromolecules of platelet membrane.

Antiplatelet Drugs and Generation of Thrombin in Clotting Blood

AbstractPlatelets participate in formation of thrombin through secretion of coagulation factors and by providing a catalytic surface on which prothrombinase complex is assembled. We studied the effects of four antiplatelet drugs on thrombin formation in healthy volunteers. Thrombin generation was monitored both in vitro—in recalcified plasma—and ex vivo—in blood emerging from a standardized skin microvasculature injury, which also served to determine bleeding time. A mathematical model has been developed to describe the latter reaction. It is based on estimation of the rate of increase in fibrinopeptide A (FPA), a specific marker of thrombin activity, in blood emerging from skin incisions. Two hours after the ingestion of 500 mg of aspirin, thrombin formation became significantly impaired both in vitro and ex vivo. In contrast, 2 hours after the oral administration of placebo, indomethacin 50 mg, or OKY-046 (a thromboxane synthase inhibitor) 400 mg, thrombinogenesis remained unaltered. Ticlopidine, studied either 3 hours after 500 mg oral administration, or after 5 days of intake at a daily dose of 500 mg, had no effect on thrombin generation. Thus, aspirin, contrary to other antiplatelet drugs, depresses thrombin formation in clotting blood, a phenomenon that might be of clinical relevance. It is suggested that aspirin exerts this effect by acetylating prothrombin and/or macromolecules of platelet membrane.

Cutaneous Blood Flow in Gestational Hypertension and Normal Pregnancy

A laser Doppler flowmeter was used to assess skin blood flow changes in three groups of young subjects: women with gestational hypertension, healthy pregnant women, and healthy non-pregnant women. Responses to four vasoactive stimuli were studied: isometric and cognitive activities, cutaneous post ischemic reactive hyperemia, and local heating. The first two stimuli are vasoconstrictive and were performed on the fingertip, whereas the latter two are vasodilative and were performed on the forearm. The most prominent differences were observed in the isometric test, where the expected decrease, which was indeed registered in non-pregnant women, was almost absent in the healthy pregnant group. The gestational hypertension group had a greater decrease in blood flow than normal pregnancy, but lesser than non-pregnant control subjects. We conclude that although normal pregnancy modifies the response of the skin microvasculature to some vasoactive stimuli, gestational hypertension pushes that response back toward the non-pregnancy state.

The quantitative contribution of nitric oxide and sensory nerves to bradykinin-induced inflammation in rat skin microvasculature

Using a blister model in the rat hind footpad, the present study undertook to examine the relative contribution of sensory nerves and nitric oxide (NO) to the inflammatory response induced by bradykinin (BK). Using this model, combined with laser Doppler flowmetry, we were able to simultaneously monitor two parameters of the inflammatory response, namely vasodilation (VD) and plasma extravasation (PE). Perfusion of BK (1, 10 or 100 μM) over the blister base elicited both VD and PE responses which were dose-dependent. The VD response was of rapid onset, sustained at the lowest concentration (1 μM), and showed tachyphylaxis at the highest two concentrations (10 and 100 μM). The PE response, however, was delayed in onset at the lower concentration but the response was maintained at all concentrations. The endothelium-independent vasodilator, sodium nitroprusside. (SNP, 100 μM), was used as an internal control and elicited a rapid maintained VD response. In rats pretreated as neonates with capsaicin to destroy primary sensory afferents, the inflammatory response to 10 μM BK was significantly smaller (50% and 64% decrease in VD and PE, respectively). The selective inhibitor of NO synthase, N G-nitro- d-arginine (D-NORAG) at 100 μM significantly attenuated the inflammatory response to BK in control rats (76% and 60% decrease in VD and PE, respectively) with a further decrease in the response in capsaicin pretreated rats. The inactive N G-nitro- d-arginine (D-NORAG) (100 μM) did not affect the inflammatory response to BK. The vasodilator response to SNP was intact in capsaicin pretreated rats and was not affected by either L-NORAG or D-NORAG. Our results suggest that sensory nerves and NO are both involved in the inflammatory response to BK in rat skin microvasculature and that a significant part of the sensory involvement is also dependent on the NO. This study is the first in vivo to provide evidence implicating NO in the inflammatory response to BK in rat skin microvasculature.

Nitric oxide and sensory nerves are involved in the vasodilator response to acetylcholine but not calcitonin gene-related peptide in rat skin microvasculature.

1. The contributions of sensory nerves and nitric oxide (NO) to vasodilator responses to acetylcholine (ACh) and calcitonin gene-related peptide (CGRP) were examined in rat skin microvasculature with a laser Doppler flowmeter to monitor relative blood flow. 2. Perfusion of ACh (100 microM; for 30 min) over a blister base on the rat hind footpad elicited microvascular vasodilatation and this response was not sustained. CGRP (1 microM; 10 min perfusion) also elicited vasodilatation and this response was maintained even when CGRP was no longer in contact with the blister base. 3. The vasodilator response to ACh was significantly smaller in rats pretreated as neonates with capsaicin to destroy primary sensory afferents than it was in age-matched controls. The vasodilator response to CGRP was unaffected by capsaicin pretreatment. 4. Selective inhibitors of NO synthase, NG-nitro-L-arginine (L-NOARG) and NG-monomethyl-L-arginine (L-NMMA) (both at 100 microM) attenuated the vasodilator response to ACh in control rats, but had no effect on the vasodilator response to CGRP. There was a significant L-NOARG-resistant component in control rats while in capsaicin-treated rats the vasodilator response to ACh was virtually abolished by L-NOARG. The inactive stereoisomer NG-monomethyl-D-arginine (100 microM) did not affect the vasodilator response to ACh. 5. The efficacy of L-NOARG and L-NMMA as inhibitors of endothelium-dependent responses was confirmed by use of an endothelium-dependent vasodilator, the calcium ionophore A23187 (100 microM; 10 min perfusion). Vasodilatation to A23187 was strongly attenuated by both L-NOARG and L-NMMA.6. These results suggest that sensory nerves and NO are both involved in the dilatation produced by ACh in rat skin microvasculature. A component of the vasodilator response elicited by ACh involves a direct action on the microvascular endothelium with subsequent generation of NO, while an additional component is elicited via activation of sensory nerves. The vasodilator mediator(s) released by ACh from sensory nerves acts largely independently of NO.7. The vasodilator response to CGRP is independent of a prejunctional action on sensory nerves and of NO.

Inflammatory effects of platelet activating factor (PAF) in equine skin.

Intradermal administration of PAF (0.001-1 micrograms/site), but not lyso-PAF (10 micrograms/site), in the horse caused an increase in cutaneous vascular permeability which was maximal by 32 min. Responses to PAF and histamine were reduced by coadministration of the histamine 1 receptor antagonist chlorpheniramine, although only the inhibition of histamine-induced responses was dose-related and statistically significant. The cyclo-oxygenase inhibitor indomethacin was without effect on PAF-induced increases in vascular permeability. These findings suggest that the actions of PAF on equine skin microvasculature may be partly due to histamine release but not to prostanoid formation. Coadministration of prostaglandin (PG) E2 enhanced the oedematous responses to both PAF and histamine, although PGE2 failed to exert direct permeability-increasing activity. In addition, and in contrast to PAF and histamine, PGE2 increased cutaneous blood flow and skin surface temperature. PAF, but not lyso-PAF, also caused neutrophil infiltration into the skin which was maximal at 2 h. No significant effects on eosinophil or mononuclear cell numbers were apparent up to 24 h after injection of PAF. These results are consistent with the concept that PAF may be a mediator of inflammatory disorders of the skin in the horse.

Chronic and Acute Effects of Cigarette Smoking on Skin Blood Flow

A laser Doppler flowmeter was used to assess blood flow changes in habitual smokers, as compared with nonsmokers, where members of both groups were young and healthy. Acute and chronic effects of cigarette smoking were measured by using the cutaneous postischemic reactive hyperemia test. Basic flow was recorded in four sites: forehead, postauricular, forearm, and finger. Recovery time from reactive hyperemia was longer in habitual smokers than in nonsmokers. Peak flow during reactive hyperemia was significantly lower after smoking. Basic blood flow during smoking did not show significant variation in the sites tested. The authors conclude that skin microvasculature is influenced by acute and chronic effects of cigarette smoking in young subjects; they discuss some of the possible mechanisms and their implications.