What is the evidence that advertising policies could have an impact on gambling-related harms? A systematic umbrella review of the literature.

OBJECTIVE: To summarise the evidence on the impacts of gambling-related advertising that could lead to gambling-related harm, including impacts on vulnerable individuals and inequalities in the distribution of harms. STUDY DESIGN: An umbrella review of studies investigating the impact of gambling advertising. METHODS: A review was undertaken of systematic reviews of qualitative, quantitative and mixed method studies reporting outcomes associated with gambling advertising and marketing. The search strategy included database searches (Web of Science, PsycInfo) and website searches. The quality of the included reviews was determined using A MeaSurement Tool to Assess systematic Reviews 2. RESULTS: 1024 papers were identified by database searches. Eight systematic reviews, including 74 unique studies, met inclusion criteria. Included studies, using quantitative and qualitative methods, consistently support the existence of a causal relationship between exposure to advertising of gambling products/brands and more positive attitudes to gambling, greater intentions to gamble and increased gambling activity at both individual and population level. There is evidence of a 'dose-response' effect; greater advertising exposure increases participation which leads to a greater risk of harm. There was more evidence for the impact on children and young people and for those already at risk from current gambling activity with those most vulnerable more likely to be influenced. CONCLUSION: Gambling advertising restrictions could reduce overall harm and mitigate the impact of advertising on gambling-related inequalities. Public health harm prevention strategies should include policies which limit exposure to advertising, particularly among children and vulnerable groups.

Arterial smooth muscle cells in vivo: relationship between actin isoform expression and mitogenesis and their modulation by heparin.

Quiescent smooth muscle cells (SMC) in normal artery express a pattern of actin isoforms with alpha-smooth muscle (alpha SM) predominance that switches to beta predominance when the cells are proliferating. We have examined the relationship between the change in actin isoforms and entry of SMC into the growth cycle in an in vivo model of SMC proliferation (balloon injured rat carotid artery). alpha SM actin mRNA declined and cytoplasmic (beta + gamma) actin mRNAs increased in early G0/G1 (between 1 and 8 h after injury). In vivo synthesis and in vitro translation experiments demonstrated that functional alpha SM mRNA is decreased 24 h after injury and is proportional to the amount of mRNA present. At 36 h after injury, SMC prepared by enzymatic digestion were sorted into G0/G1 and S/G2 populations; only the SMC committed to proliferate (S/G2 fraction) showed a relative slight decrease in alpha SM actin and, more importantly, a large decrease in alpha SM actin mRNA. A switch from alpha SM predominance to beta predominance was present in the whole SMC population 5 d after injury. To determine if the change in actin isoforms was associated with proliferation, we inhibited SMC proliferation by approximately 80% with heparin, which has previously been shown to block SMC in late G0/G1 and to reduce the growth fraction. The switch in actin mRNAs and synthesis at 24 h was not prevented; however, alpha SM mRNA and protein were reinduced at 5 d in the heparin-treated animals compared to saline-treated controls. These results suggest that in vivo the synthesis of actin isoforms in arterial SMC depends on the mRNA levels and changes after injury in early G0/G1 whether or not the cells subsequently proliferate. The early changes in actin isoforms are not prevented by heparin, but they are eventually reversed if the SMC are kept in the resting state by the heparin treatment.

Local overexpression of TIMP-1 prevents aortic aneurysm degeneration and rupture in a rat model.

Although matrix metalloproteinases (MMPs) are expressed in abundance in arterial aneurysms, their contribution to arterial wall degeneration, dilation, and rupture has not been determined. We investigated MMP function in a rat model of aneurysm associated with arterial dilation, elastin loss, medial invasion by mononuclear inflammatory cells, and MMP upregulation. Rupture was correlated with increased gelatinase B (MMP-9) and activated gelatinase A (MMP-2). Syngeneic rat smooth muscle cells retrovirally transfected with tissue inhibitor of matrix metalloproteinases (TIMP)-1 cDNA (LTSN) or with the vector alone as a control (LXSN) were seeded onto the luminal surface of the vessels. The seeding of LTSN cells resulted in TIMP-1 local overexpression. The seeding with LTSN cells, but not LXSN cells, decreased MMP-9, activated MMP-2 and 28-kD caseinase and elastase activity, preserved elastin in the media, and prevented aneurysmal degeneration and rupture. We conclude that MMP overexpression is responsible for aneurysmal degeneration and rupture in this rat model and that local pharmacological blockade might be a reasonable strategy for controlling the formation of aneurysms in humans.

Long-term biological response of injured rat carotid artery seeded with smooth muscle cells expressing retrovirally introduced human genes.

Cultured vascular smooth muscle cells (SMCs) containing retrovirally introduced genes are a potential vehicle for gene replacement therapy. Because the cultured SMCs are selected for their ability to proliferate in vitro, it is possible that the SMCs might be permanently altered and lose their capacity to respond to growth-suppressing conditions after being seeded back into blood vessels. To investigate this possibility we measured SMC proliferation and intimal thickening in balloon-injured Fischer 344 rat carotid arteries seeded with SMCs stained with the fluorescent marker 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate (DiI) and infected with replication-defective retrovirus expressing human adenosine deaminase or human placental alkaline phosphatase. The majority of the seeded SMCs remained in the intima while a few of the cells appeared to migrate into the first layer of the media. Intimal SMC proliferation returned to background levels (< 0.1% thymidine labeling index) by 28 d. At late times (1 and 12 mo) the morphological appearance of the intima was the same for balloon-injured arteries with or without seeded SMC, except that the seeded arteries continued to express human adenosine deaminase or alkaline phosphatase. These results support the conclusion that cultured SMC infected with a replication-defective virus containing human adenosine deaminase or alkaline phosphatase are not phenotypically altered and do not become transformed. After seeding onto the surface of an injured artery, they stop replicating but continue to express the introduced human genes even over the long term.

Mitomycin C augmented glaucoma surgery: evolution of filtering bleb avascularity, transconjunctival oozing, and leaks.

AIMS: To prospectively study the evolution of possible high risk features of conjunctival filtration blebs like avascularity, transconjunctival oozing (TCO), and leaks after mitomycin C (MMC) enhanced glaucoma surgery. METHODS: Single observer, 2 year prospective study on bleb characteristics of 125 eyes of 125 consecutive patients who had MMC augmented glaucoma surgery with initially successful filtration. MMC (0.2 mg/ml for 2 minutes in most cases) was applied on the area of the scleral flap before dissection. Glaucoma surgeries included were trabeculectomy, deep sclerectomy, and combined procedures. A dry fluorescein strip was applied on the avascular part of the bleb and observed for aqueous egress with flow (point leak, PL) or without (TCO). RESULTS: The mean time (95% CI) for observing bleb avascularity, TCO, and bleb leaks were 106 days (69 to 143), 208 days (155 to 261), and 609 days (559 to 659), respectively. Bleb leaks were observed in 17 eyes (13.6%)-15 (24.6%) in the trabeculectomy group and two (3.1%) in the deep sclerectomy group (p = 0.003). Kaplan-Meier survival analyses showed that the probability of observing bleb avascularity at sixth, 12th, and 24th month after surgery was 56%, 71%, and 73%, respectively. In eyes with avascular blebs, the probability of developing TCO and leaks was 77% and 1% at 6 months, 81% and 12% at 12 months, and 95% and 26% at 24 months, respectively. Cox's regression analyses and log rank tests showed that eyes with larger avascular blebs (>4 mm) were more likely to develop TCO (hazard ratio 3.77, p = 0.001) and delayed bleb leaks were more likely to be seen in eyes of the trabeculectomy group rather than the deep sclerectomy group (hazard ratio 0.06, p = 0.0006). CONCLUSIONS: MMC application over the area of scleral flap dissection during glaucoma surgery is associated with a high incidence of bleb avascularity, TCO, and delayed bleb leaks. Most eyes developed bleb avascularity within the first year after surgery. TCO will eventually be seen in all eyes with avascular blebs and the incidence of leaks gradually increases with time. This study shows that patients with eyes undergoing glaucoma surgery with MMC and avascular blebs should be monitored indefinitely.

Cultured arterial smooth muscle cells maintain distinct phenotypes when implanted into carotid artery.

Cultured arterial smooth muscle cells (SMCs) with distinct phenotypic features have been described by several laboratories; however, it is not presently known whether this phenotypic heterogeneity can be maintained within an in vivo environment. To answer this question, we have seeded into the intima of denuded rat carotid artery 2 SMC populations with well-established distinct biological features, ie, spindle-shaped, not growing in the absence of serum, and well differentiated versus epithelioid, growing in the absence of serum, and relatively undifferentiated, derived from the aortic media of newborn rats (aged 4 days) and old rats (aged >18 months), respectively. We show that these 2 populations maintain their distinct biochemical features (ie, expression of alpha-smooth muscle actin, smooth muscle myosin heavy chains, and cellular retinol binding protein-1) in the in vivo environment. The old rat media-derived SMCs continue to produce cellular retinol binding protein-1 but little alpha-smooth muscle actin and smooth muscle myosin heavy chains, whereas the newborn rat media-derived SMCs continue to express alpha-smooth muscle actin and smooth muscle myosin heavy chains but no cellular retinol binding protein-1. Our results reinforce the notion of arterial SMC phenotypic heterogeneity and suggest that in our model, heterogeneity is controlled genetically and not by the local environment.

Combined cataract and glaucoma surgery with mitomycin C: phacoemulsification-trabeculectomy compared to phacoemulsification-deep sclerectomy.

AIMS: To compare outcomes of phacoemulsification combined with trabeculectomy (PT) or deep sclerectomy (PDS) with intraoperative mitomycin C (MMC) application. METHODS: Non-randomised, consecutive, retrospective comparative study. 97 eyes of 97 patients (59 PDS, 38 PT) undergoing combined surgery with intraoperative MMC (0.1-0.4 mg/ml for 1-3 minutes) were identified for inclusion in the study. RESULTS: The probability of maintaining intraocular pressure (IOP) below 19 mm Hg and 15 mm Hg, with a 30% drop from preoperative IOP and without additional medication, 1 year after surgery were 77.6% (95% CI: 67 to 90) and 71.5% (60 to 85) for the PDS group and 89.5% (80 to 99) and 89.5 (80 to 99) for the PT group, respectively, and these differences were not statistically significant (p>0.05, log rank test). After excluding ocular co-morbidity no differences were observed in the improvement of visual acuity between the two groups. There were no major differences in the complication rates except that delayed bleb leaks were seen in seven eyes (18.4%) of the PT group (p = 0.004). CONCLUSION: In this study, no statistically significant difference was found in the IOP and visual outcomes between PDS and PT. A significantly higher frequency of late bleb leaks after PT was observed.

Heparin and cilazapril together inhibit injury-induced intimal hyperplasia.

Both heparin and the angiotensin converting enzyme inhibitor cilazapril inhibit intimal thickening in rat carotid arteries injured by the passage of a balloon catheter. The purpose of this study was to determine if combinations of the two drugs were more effective than either drug alone and whether the effect could be accounted for by inhibition of smooth muscle cell proliferation. Heparin (0.1-0.3 mg/kg/hr) administered by continuous intravenous infusion with or without cilazapril (0-25 mg/kg/day p.o.) produced a dose-dependent inhibition of smooth muscle accumulation at 14 days after rat carotid ballooning. At the lower doses, the inhibitory effects of heparin and cilazapril were additive when the drugs were used together. This overall effect on growth was reflected in decreased smooth muscle cell proliferation at 2 and 7 days. A 7-day course of heparin combined with cilazapril, a regimen that might be applicable in the clinical setting, produced an 80% inhibition of intimal thickening at 28 days. These results provide evidence that heparin and cilazapril together might prove to be more effective than either drug alone in the control of intimal hyperplasia after arterial injury.

Generating antibodies against secreted proteins using vascular smooth muscle cells transduced with replication-defective retrovirus.

The traditional method of antibody (Ab) generation requires repeated injections of antigen (Ag). We have developed an alternative method that allows an investigator to generate a polyclonal antiserum with only a cDNA in hand. We cloned a cDNA encoding the coding frame for baboon tissue inhibitor of matrix metalloproteinase-1 (TIMP-1). Fischer rat arterial smooth muscle cells (SMC) transduced with the baboon TIMP-1 using a replication-defective retrovirus were propagated in culture. TIMP-1 overexpressing rat SMC were seeded into de-endothelialized rat carotid arteries. Three weeks after cell seeding in the rat, the presence of Ab to the baboon TIMP-1 was detected by dot blot and enzyme-linked immunosorbent assay in 5 of 6 of the animals. The major portion of the Ab generated against baboon TIMP-1 during the 12-month monitoring period after the cell seeding was identified as belonging to the IgG1 subtype. More interestingly, the titer of the Ab kept rising throughout an 8-month monitoring period. Among the salient features of this Ab are its capacity to block TIMP-1 activity and its utility for detecting TIMP-1 by immunohistochemistry. These results demonstrate that Ab against a secreted protein can be obtained in response to continuous expression of the cDNA by vascular SMC. Purified Ag is not required.

Evaluation of the Honeywell ACS 1000 leucocyte differential counter.

The Honeywell ACS 1000 is a relatively inexpensive differential white cell counter, which is only partially automated. This instrument has been evaluated in a routine haematology laboratory.

The influence of hypertension on injury-induced myointimal thickening.

Although hypertension has been identified as a risk factor in atherosclerosis, how hypertension enhances plaque growth is not clear. To study the influence of essential hypertension on injury-induced arterial intimal thickening, we employed a model of arterial endothelial injury (aortic balloon injury) in spontaneously hypertensive rats (SHR). SHR rendered normotensive with drugs served as controls. The injured vessels were fixed by perfusion at intervals between 2 weeks and 3 months and studied by light, transmission, and scanning electron microscopy. Endothelial regeneration at 2 weeks was assessed by the difference between total and blue-stained arterial surface area in rats receiving Evans blue by injection and was decreased in SHR. Intimal thickening was increased in SHR as compared with controls at all time intervals and appeared to be due to increased smooth muscle cell proliferation. Although neither SHR or controls injured arteries were stained by Evans blue at 3 months, the SHR (but not the control) injured arteries demonstrated subendothelial edema and focal necrosis in the intima. These data in a model of arterial endothelial injury support the concept that essential hypertension has a deleterious effect on arterial wound healing by enhancing arterial myointimal thickening. This effect can be reduced by adequate control of the hypertension with drugs.

Regulation of smooth muscle proliferation by heparin in vitro and in vivo.

Smooth muscle cell proliferation is central to the development of atherosclerotic plaques, intimal thickening, and recurrent stenosis in arteries following surgical reconstruction. The factors that might limit this process remain poorly defined. Studies reported recently from several laboratories suggest that heparin administered in pharmacological doses can suppress proliferation of smooth muscle cells. Furthermore, heparin-like molecules are synthesized by vascular wall cells, inhibit smooth muscle growth in vitro, and might act to regulate smooth muscle growth within the arterial wall.

Kinetics of cellular proliferation after arterial injury. II. Inhibition of smooth muscle growth by heparin.

Heparin inhibits intimal thickening after arterial injury. Whether this effect is due to inhibition of medial smooth muscle cell (SMC) migration, SMC proliferation in the intima, or synthesis and deposition of connective tissue has not been evident. In this study we have investigated these possibilities in a rat carotid balloon injury model. Heparin (0.3 mg/kg/hour) was administered intravenously by means of osmotic pumps to experimental animals, and controls received lactated Ringer's solution. Smooth muscle proliferation (thymidine index), intimal smooth muscle accumulation, and endothelial regeneration were measured at intervals between 0 and 28 days. Total smooth muscle growth as determined biochemically at 14 days was markedly inhibited by heparin if the pumps were placed 24 hours before or at the time of injury and less so if inserted 48 or 96 hours after injury. SMC thymidine indices were maximal in the media at 4 days and in the intima at 7 days for injured arteries of both heparin-treated and control rats; at each time point SMC proliferation and intimal thickening were less in heparin-treated rats. The volume of connective tissue in the intima was the same in both groups at 28 days. Medial SMC migration into the intima was diminished by heparin treatment, but endothelial regeneration was not affected. These results support the hypothesis that heparin is a specific inhibitor of SMC migration and proliferation and is most effective if started before SMC enter S-phase.

Influence of chronic hypertension on injured and uninjured arteries in spontaneously hypertensive rats.

The influence of chronic essential hypertension in spontaneously hypertensive rats (SHR) on uninjured and deendothelialized arteries was investigated in two models of arterial endothelial injury (aortic balloon and carotid air injury). SHR rendered normotensive with antihypertensive medication (hydralazine, reserpine, and furosemide), normotensive Wistar rats, and Wistar rats treated with antihypertensive drugs served as controls. In uninjured vessels, slight intimal thickening and marked medial thickening were found in SHR but not controls. In injured vessels, endothelial regeneration was similar in SHR and controls. Intimal thickening due to smooth muscle cell proliferation was markedly enhanced in SHR compared to drug-treated SHR, whereas medial thickening in the injured vessels of SHR did not change over the time interval studied. Administration of drug to normotensive Wistar rats did not affect myointimal or medial thickening. These results indicate that acute arterial endothelial injury in chronically hypertensive rats produces marked myointimal thickening that can be controlled with antihypertensive medication.

Kinetics of cellular proliferation after arterial injury. IV. Heparin inhibits rat smooth muscle mitogenesis and migration.

Heparin inhibits the development of intimal thickening after carotid injury in the rat; however, the specific cellular events responsible for this effect have not been defined. In this study, smooth muscle cell growth fraction and migration into the intima were quantitatively measured in heparin-treated animals. All rats were subjected to left carotid balloon injury and received continuous intraperitoneal infusion of tritiated thymidine; they were given either heparin or lactated Ringer's solution intravenously for periods of time up to 7 days. Both smooth muscle cell growth fraction and migration of nondividing smooth muscle cells were markedly reduced in heparin-treated rats. If heparin was administered for only the first 3 days after carotid injury, both smooth muscle growth fraction and migration were reduced at 7 days; on the other hand, heparin had no effect on growth fraction and migration if given from day 4 to day 7. Finally, heparin given for a period of 1 week produced marked reduction in smooth muscle cell accumulation in injured arteries at 2 and 4 weeks. These results suggest that the effect of heparin on injury-induced intimal thickening might be due to inhibition of both smooth muscle cell entry into the growth fraction and migration of medial smooth muscle cells into the intima. These effects are long lasting, and are not reversed even if heparin is stopped after a short course of administration.

Kinetics of cellular proliferation after arterial injury. III. Endothelial and smooth muscle growth in chronically denuded vessels.

The healing of injured rat carotid chronically denuded of endothelium was studied at late times after balloon injury. One year after injury the endothelial layer was not fully regenerated and had ceased proliferating. The denuded areas of these vessels were covered with luminal smooth muscle cells which strongly resembled endothelium but did not stain with factor VIII.R.Ag antibody. These cells were actively proliferating and formed a surface which was weakly thrombogenic. Indium-111 platelet studies showed that over 24 hours there was a slight but significant adherence of platelets to the denuded surface. Luminal smooth muscle cell proliferation was matched by cell loss and did not produce further intimal thickening or an increase in total cell number. These results demonstrate that damaged conduit arteries can exist in a stable state without endothelium and do not develop thrombosis even after prolonged periods of time.

Mechanisms of stenosis after arterial injury.

The development of luminal narrowing in relationship to intimal thickening was investigated in rat common carotid artery denuded of endothelium. After denudation with a balloon embolectomy catheter endothelium was observed to regenerate from the ends of the denuded segment but not to cover the central third of the artery by 12 weeks. Cross-sections of denuded (Evans blue stained) and reendothelialized (white) areas showed that intimal thickening in the blue, but not the white, region progressively increased with time and was maximal between 4 and 12 weeks. Luminal narrowing was most pronounced at 2 weeks (75%) and less at 12 weeks (35%). The apparent discrepancy in these results was resolved by demonstrating that the vessel circumference in the blue region at the level of the internal elastic lamina was reduced at 2 weeks and the same as controls at 12 weeks; intravenous infusion of papaverine abolished this vasoconstriction of the left carotids at 2 weeks after injury. These results demonstrate that luminal narrowing early after injury is in large part due to smooth muscle contraction of the vessel and late due only to intimal thickening. During the period between 2 and 12 weeks the fraction of the intima occupied by smooth muscle cells decreased markedly, but the total volume due to smooth muscle cells remained relatively constant. Previous studies have demonstrated by DNA measurements that arterial wall cell number is the same at 2 and 12 weeks; these results taken together indicate that continued intimal thickening at late time points is due to synthesis and accumulation of connective tissue without further increase in smooth muscle cell number.

Kinetics of cellular proliferation after arterial injury. I. Smooth muscle growth in the absence of endothelium.

Intimal smooth muscle (SMC) proliferation was examined in the rat left carotid in regions lacking endothelium for prolonged periods of time. Arteries of animals injected with tritiated thymidine and Evans blue were examined at intervals between 0 and 12 weeks. The endothelial layer was regenerated from the ends of the denuded segment but failed to cover the central third of the artery by 12 weeks. Autoradiography on samples from this central region (stained blue) and the endothelialized ends (white) showed that SMC proliferation reached a maximum at 48 hours in the media (46%) and at 96 hours in the intima (73%). Subsequently, the thymidine index declined to baseline (0.06%) by 4 weeks throughout the media and by 8 weeks in the intima covered by endothelium. SMC proliferation persisted at a high level (3.8%) at the surface of the intima lacking endothelium even at 12 weeks. Despite continued proliferation of luminal SMC, total arterial SMC number was the same at 2 and 12 weeks. These results support the concept that intimal SMC proliferation after arterial injury is an acute event related to the initial injury process. Persistent proliferation of luminal SMC does not result in an increase in intimal cell number.

Mechanisms of arterial graft failure. II. Chronic endothelial and smooth muscle cell proliferation in healing polytetrafluoroethylene prostheses.

In a previous study of arterial bypass grafts (4 mm polytetrafluoroethylene [PTFE]) in baboons we observed that endothelial and smooth muscle cells (SMCs) formed the neointima and were derived from the cut edges of adjacent artery. The purpose of this study was to determine at late times whether endothelial cells would continue to migrate and to proliferate to cover the graft surface and whether the underlying proliferating SMCs would produce a progressively thickened intima, graft stenosis, and eventual thrombosis. At 6 and 12 months after grafts were placed, endothelial coverage by ingrowth from the anastomoses was more advanced than at 3 months, and by 12 months 60% of grafts (7 to 9 cm in length) were covered. Endothelial cells proliferated in association with the growing edge and focally in other regions. Underlying SMCs proliferated in the region of the growing edge of the endothelial cells and also at anastomoses. Intimal cross-sectional area was greatest at anastomoses and at late times was principally due to an increase in connective tissue; actual SMC mass remained constant after 3 months. These results demonstrated slow but progressive healing of the grafts by ingrowth of endothelium. There was also an increased turnover rate of SMCs and endothelial cells in established intima at late times, which might be the consequence of chronic endothelial injury. This condition represents a stable state since it does not produce further intimal thickening and accumulation of SMCs and does not lead to a high rate of thrombosis.